Compositions and methods for producing dendritic cells

ABSTRACT

Described herein are compositions and methods for treating a disease, particularly a cancer, with primed dendritic cells recognizing a tumor antigen. The methods may comprise storing, shipping and/or culturing dendritic cells, where the dendritic cells are stored on a hard surface. Lysis protocols are described where the lysis does not result in complete lysis of cells in order to provide cell surface molecules maintained in a cell surface-embedded state. Non-lethal Dengue virus strains are also provided for therapeutic purposes.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.62/284,434, filed Sep. 26, 2015, which is incorporated herein byreference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 22, 1016, isnamed 48253-703_201_SL.txt and is 1831 bytes in size.

BACKGROUND

Dendritic cells (DCs) are antigen-presenting cells of the immune system.They engulf and process bits of bacteria, viruses, and other pathogensbefore presenting the relevant protein chain targets (antigenicpeptides), to Cytotoxic T Lymphocytes (CTL), which recognize and killvirus-infected or cancer cells, and B-lymphocytes, which makeantibodies. DCs also engulf cells which are damaged or dead, and arerequired to induce either a Type 1 response (activation), a Type 2response (tolerant), or a Type 0 response (neutral). Because the same 20amino acids make up body parts (self), as well as pathogens (non-self),DCs must evaluate not only the antigen structure, but also the cytokineand other signaling environment present at the time. This multi-layeredsystem is in place to prevent auto-immunity, where the immune systemmistakes self for non-self, as well as allergic responses, where aneutral response is required to maintain balance. This complex system ofinternal checks and balances is exploited by tumor cells, which arisefrom “self” cells. Tumor cells often secrete factors such asTransforming Growth Factor-beta (TGFβ) which switch responding immunecells toward a T_(H)2-type tolerant response. This allows the tumorcells to grow unchecked, and often aided by, the immune cells. DCs havethe capability of programming CD4⁺ and CD8⁺ CTL to recognize the MHC(self-protein ID complex) and associated peptide presented. However, theCTL must then decide whether to ignore the cell as self, or initiatelysis, e.g., through the Fas/FasL or Perforin/Granzyme B cell-deathsystems. The decision will often rely on the activation state of theCTL, the cytokine environment, and the presence or absence ofcell-damage factors, e.g., heat-shock proteins, Toll-like receptoractivation signals, and the like. During an active pathogen infection,these systems become activated and help steer the CTL response to a Type1 attack mode.

Immunotherapy, unlike cytotoxic drugs, radiation, and surgery,stimulates the immune system to recognize and kill tumor cells. Numerousattempts have been made in stimulating the immune system to recognizeand destroy tumor cells. These have been met with limited success due tothe self-identity of peptides selected as target for immunotherapy, lackof immune activation, adverse events, and/or tumor immune evasionmechanisms.

The ability of current cellular therapies, e.g., dendritic celltherapies, to induce durable, complete responses in advanced cancerpatients is low (5-10% in the most immunogenic cancer types, lower inothers). Often, dendritic cell therapies produce less than desirableresults because of low activation (e.g. not enough immune cells toadequately kill all cancer cells), low targeting (e.g., healthy cellsare killed and/or tumor cells are not killed), or an immunosuppressedtumor microenvironment, limiting drug efficacy.

BRIEF SUMMARY

Provided herein are methods for producing primed dendritic cells,comprising: culturing dendritic cells on a hard surface; lysing at leastone cell with a hypochlorite solution to produce a lysate; contactingthe dendritic cells with the lysate to produce primed dendritic cells;and maturing the primed dendritic cells, wherein maturing comprisescontacting the primed dendritic cells with a toll-like receptor 7agonist or a toll-like receptor 8 agonist. Further provided herein aremethods, wherein the primed dendritic cells produce at least 6 ng/mLIL-12p70. Further provided herein are methods, wherein the primeddendritic cells produce at least about 6.5 ng/mL IL-12p70. Furtherprovided herein are methods, wherein the primed dendritic cells produceabout 19 ng/mL IL-12p70. Further provided herein are methods, whereinthe hard surface is a plastic surface. Further provided herein aremethods, wherein the hard surface comprises polystyrene. Furtherprovided herein are methods, wherein the hard surface is substantiallyfree of a component that reduces a Type 1 response produced by theprimed dendritic cells. Further provided herein are methods, wherein thecomponent is selected from a fluorinated polyethylene, a fluorinatedpolypropylene, and a phthalate. Further provided herein are methods,wherein the lysate comprises a plurality of intact cells. Furtherprovided herein are methods, wherein the toll-like receptor 7 agonist ortoll-like receptor 8 agonist is an imidazoquinoline compound. Furtherprovided herein are methods, wherein the imidazoquinoline compound isR848. Further provided herein are methods, wherein maturing comprisescontacting the primed dendritic cells with (i) a toll-like receptor 2agonist or a toll-like receptor 4 agonist, or (ii) interferon gamma.Further provided herein are methods, wherein the toll-like receptor 2agonist or the toll-like receptor 4 agonist is lipopolysaccharide.Further provided herein are methods, wherein maturing further comprisescontacting the primed dendritic cells with (i) a toll-like receptor 2agonist or a toll-like receptor 4 agonist; and (ii) interferon gamma.Further provided herein are methods, wherein maturing comprisescontacting the primed dendritic cells with R848; lipopolysaccharide; andinterferon gamma. Further provided herein are methods, wherein the atleast one cell is a tumor cell. Further provided herein are methods,wherein the dendritic cells are autologous or allogeneic to the subject.Further provided herein are methods, wherein the dendritic cells areallogeneic cells that are HLA matched to the subject. Further providedherein are methods, comprising obtaining the at least one cell from asubject, wherein the cell is associated with a harmful disease state.Further provided herein are methods, wherein the harmful disease stateis a proliferative disorder or an autoimmune disorder. Further providedherein are methods, wherein maturing further comprises contacting theprimed dendritic cells with (i) a toll-like receptor 2 agonist, atoll-like receptor 4 agonist, or (ii) interferon gamma. Further providedherein are methods, wherein the toll-like receptor 2 agonist or thetoll-like receptor 4 agonist is lipopolysaccharide. Further providedherein are methods, wherein maturing further comprises contacting theprimed dendritic cells with (i) a toll-like receptor 2 agonist or atoll-like receptor 4 agonist; and (ii) interferon gamma. Furtherprovided herein are methods, wherein maturing comprises contacting theprimed dendritic cells with R848; lipopolysaccharide; and interferongamma. Further provided herein are methods, wherein a population of theprimed dendritic cells has a viability of greater than 70% afterfreezing and thawing. Further provided herein are methods, wherein apopulation of the primed dendritic cells has a viability of about 71% toabout 79% after freezing and thawing.

Provided herein are methods for producing primed dendritic cells,comprising: culturing dendritic cells on a hard surface; obtaining atleast one cancer cell from a subject; lysing the at least one cancercell with a hypochlorite solution to produce a lysate; contacting thedendritic cells with the lysate to produce primed dendritic cells; andmaturing the primed dendritic cells, wherein maturing comprisescontacting the primed dendritic cell with a toll-like receptor 7 agonistor a toll-like receptor 8 agonist. Further provided herein are methods,wherein the primed dendritic cells produce at least 6 ng/mL IL-12p70.Further provided herein are methods, wherein the primed dendritic cellsproduce at least about 6.5 ng/mL IL-12p70. Further provided herein aremethods, wherein the primed dendritic cells produce about 19 ng/mLIL-12p70. Further provided herein are methods, wherein the hard surfaceis a plastic surface. Further provided herein are methods, wherein thehard surface comprises polystyrene. Further provided herein are methods,wherein the hard surface is substantially free of a component thatreduces a Type 1 response produced by the primed dendritic cells.Further provided herein are methods, wherein the component is selectedfrom a fluorinated polyethylene, a fluorinated polypropylene, and aphthalate. Further provided herein are methods, wherein the lysatecomprises a plurality of intact cells. Further provided herein aremethods, wherein the plurality of intact cells is a plurality ofproliferation-inactivated cancer cells. Further provided herein aremethods, wherein the toll-like receptor 7 agonist or toll-like receptor8 agonist is an imidazoquinoline compound. Further provided herein aremethods, wherein the imidazoquinoline compound is R848. Further providedherein are methods, wherein the toll-like receptor 2 agonist or atoll-like receptor 4 agonist is lipopolysaccharide. Further providedherein are methods, wherein the dendritic cells are autologous orallogeneic to the subject. Further provided herein are methods, whereinthe dendritic cells are allogeneic cells that are HLA matched to thesubject. Further provided herein are methods, wherein maturing furthercomprises contacting the primed dendritic cells a toll-like receptor 2agonist, a toll-like receptor 4 agonist, interferon gamma, orcombinations thereof.

Provided herein are methods for treatment or reduction of a cancer in asubject in need thereof, comprising: culturing dendritic cells on a hardsurface; obtaining at least one cancer cell; lysing the at least onecancer cell with a hypochlorite solution to produce a lysate; contactingthe dendritic cells with the lysate, thereby generating primed dendriticcells; and administering the primed dendritic cells to a subject in needthereof. Further provided herein are methods, wherein the primeddendritic cells produce at least about 6 ng/mL IL-12p70. Furtherprovided herein are methods, wherein the primed dendritic cells produceat least about 6.5 ng/mL IL-12p70. Further provided herein are methods,wherein the primed dendritic cells produce about 19 ng/mL IL-12p70.Further provided herein are methods, wherein the primed dendritic cellsproduce 19 ng/mL IL-12p70. Further provided herein are methods, whereinthe primed dendritic cells produce about 15 ng/mL IL-12p70 to about 23ng/mL IL-12p70. Further provided herein are methods, wherein the primeddendritic cells produce 15 ng/mL IL-12p70 to 23 ng/mL IL-12p70. Furtherprovided herein are methods, wherein the dendritic cells are autologousor allogenic to the subject. Further provided herein are methods,wherein the at least one cancer cell is from the subject. Furtherprovided herein are methods, comprising maturing the primed dendriticcells, wherein maturing comprises adding a maturation reagent, whereinthe maturation reagent comprises a toll-like receptor 7 agonist or atoll-like receptor 8 agonist. Further provided herein are methods,wherein maturing further comprises contacting the primed dendritic cellswith (i) a toll-like receptor 2 agonist or a toll-like receptor 4agonist; or (ii) interferon gamma. Further provided herein are methods,wherein the toll-like receptor 2 agonist or the toll-like receptor 4agonist is lipopolysaccharide. Further provided herein are methods,wherein maturing further comprises contacting the primed dendritic cellswith (i) a toll-like receptor 2 agonist or a toll-like receptor 4agonist; and (ii) interferon gamma. Further provided herein are methods,wherein maturing comprises contacting the primed dendritic cells withR848; lipopolysaccharide; and interferon gamma.

Provided herein are methods for treatment or reduction of cancer in asubject in need thereof, comprising: obtaining dendritic cells from asubject; culturing the dendritic cells on a hard surface; obtaining acancer cell from the subject; lysing the cancer cell with a hypochloritesolution to produce a lysate; contacting the dendritic cells with thelysate, thereby generating primed dendritic cells; administering theprimed DCs to the subject; and administering a Dengue virus to thesubject in need thereof. Further provided herein are methods, whereinthe Dengue virus is DENV-2 strain #1710. Further provided herein aremethods, wherein the hard surface is a hard plastic surface. Furtherprovided herein are methods, wherein the hard plastic surface is apolystyrene surface. Further provided herein are methods, wherein theprimed dendritic cells produce at least 6 ng/mL IL-12p70. Furtherprovided herein are methods, wherein the primed dendritic cells produceat least about 6.5 ng/mL IL-12p70. Further provided herein are methods,wherein the primed dendritic cells produce about 19 ng/mL IL-12p70.Further provided herein are methods, wherein the primed dendritic cellsproduce about 6.5 to 23 ng/mL IL-12p70.

Provided herein are methods for clearing cancer cells in a subject,comprising: administering a Dengue virus serotype 2 to a subject in needthereof; priming a dendritic cells, wherein priming comprises: exposingthe dendritic cells to a lysate to produce primed dendritic cells,wherein the lysate comprises a plurality of cancer cells, each cancercell comprising an antigen present on the surface of said cancer cell;and administering the primed dendritic cells to the subject, wherein theadministration provides for clearance of 33% or more of a cancer cellpopulation in the subject. Further provided herein are methods whereinthe administration provides for clearance of 33% of the cancer cellpopulation in the subject. Further provided herein are methods, furthercomprising intravenously administering the Dengue virus serotype 2 andintravenously administering the population of primed dendritic cells.Further provided herein are methods, wherein the plurality of cancercells is from the subject. Further provided herein are methods, whereinthe Dengue virus serotype 2 is DENV-2 strain #1710.

Provided herein are methods for treating or reducing cancer in a subjectin need thereof, comprising administering the primed dendritic cellsproduced by a method comprising: culturing dendritic cells on a hardsurface; lysing at least one cell with a hypochlorite solution toproduce a lysate; contacting the dendritic cells with the lysate toproduce primed dendritic cells; and maturing the primed dendritic cells,wherein maturing comprises contacting the primed dendritic cell with atoll-like receptor 7 agonist or a toll-like receptor 8 agonist. Furtherprovided herein are methods, wherein the primed dendritic cells areadministered intravenously. Further provided herein are methods,comprising administering Dengue Virus 2 to the subject. Further providedherein are methods, wherein the Dengue Virus 2 is strain #1710.

Provided herein are primed dendritic cells produced by a methodcomprising: culturing dendritic cells on a hard surface; lysing at leastone cell with a hypochlorite solution to produce a lysate; contactingthe dendritic cells with the lysate to produce primed dendritic cells;and maturing the primed dendritic cells, wherein maturing comprisescontacting the primed dendritic cell with a toll-like receptor 7 agonistor a toll-like receptor 8 agonist. Further provided herein are cells,wherein the primed dendritic cell is used for treatment of a cancer.

Provided herein are uses of the primed dendritic cells prepared by amethod comprising: culturing dendritic cells on a hard surface; lysingat least one cell with a hypochlorite solution to produce a lysate;contacting the dendritic cells with the lysate to produce primeddendritic cells; and maturing the primed dendritic cells, whereinmaturing comprises contacting the primed dendritic cell with a toll-likereceptor 7 agonist or a toll-like receptor 8 agonist, for treatingcancer.

Provided herein are effective amounts of the primed dendritic cellsprepared by a method comprising: culturing dendritic cells on a hardsurface; lysing at least one cell with a hypochlorite solution toproduce a lysate; contacting the dendritic cells with the lysate toproduce primed dendritic cells; and maturing the primed dendritic cells,wherein maturing comprises contacting the primed dendritic cell with atoll-like receptor 7 agonist or a toll-like receptor 8 agonist, for usein treating cancer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts an exemplary method of treatment with Dengue virus andprimed dendritic cells.

FIG. 2 exemplifies a method of treatment with Dengue virus and primeddendritic cells schedule for treatment with Dengue virus and primeddendritic cells

FIG. 3 shows a plot of corresponding to the number of lung metastasesfrom melanoma cells in mice under various treatment conditions. Thepatterned bars represent the mean number of lung metastases for eachcondition.

FIG. 4 shows a plot of corresponding to the number of lung metastasesfrom melanoma cells in mice under various treatment conditions. Thepatterned bars represent the mean number of lung metastases for eachcondition.

FIG. 5 shows a plot of flow cytometry data confirming isolation of CD14+monocytes.

FIG. 6 shows a chart representing protein expression of IL-12p70 by DCsproduced by methods disclosed herein relative to that of DCs produced byseveral comparator methods.

DETAILED DESCRIPTION

Provided herein are methods for preparation of primed dendritic cells(DCs). Further provided herein are methods for exposing the primeddendritic cells to antigens associated with a disease state, e.g., tumorantigens, resulting primed dendritic cells capable of inducing specificand robust responses from cytotoxic T lymphocyte (CTL) toward cancercells. Further provided herein are methods for administering such DCsinto a subject for treatment of a disorder linked to the disease state.In some instances, the disorder is cancer. In some instances, thedisorder is an autoimmune disorder, e.g., rheumatoid arthritis andmultiple sclerosis. In some instances, the disorder is a humanimmunodeficiency virus (HIV) infection or an acquired immunodeficiencysyndrome. In some instances, the subject is administered a Dengue Virusprior to administration of the primed DCs.

Priming the dendritic cells generally involves contacting the dendriticcells with one or more tumor antigens that are present on target cancercells. In some cases, the dendritic cells are primed with the tumorantigen alone, the tumor antigen having been synthesized, isolated orpurified. Alternatively or additionally, the dendritic cells are primedwith a tumor cell lysate, wherein the tumor cell lysate contains thetumor antigen. In some cases, the dendritic cell is primed with a wholecancer cell expressing the tumor antigen. The dendritic cell is thenadministered to the subject, where it will present the tumor antigen tothe CTL, and thus, tailor the CTL for recognition and destruction oftarget cancer cells.

Provided herein are methods which limit dendritic cells exposure topolymers contained in plastic container material. For example, in thecase of soft plastic bags, polymers may leach into the media solutionand impact DC activity. Instead, dendritic cells may be cultured, storedand shipped in and on a hard container, such as a polystyrene tissueculture plate. This avoids a reduction in dendritic cellimmunostimulatory activity that can be caused by exposure to polymerscontained in soft plastic bags. For example, these polymers can reducethe amount of IL-12 produced by the dendritic cells, thereby reducingtheir capacity to induce a robust CTL response. Examples provided hereindemonstrate that primed dendritic cells generated by the methodsdisclosed herein are capable of secreting at least 18 pg/mL of IL-12p70,whereas dendritic cells produced by standard methods typically onlyproduce 4-6 pg/mL of IL-12p70.

In some cases, it is desirable or advantageous to prime the dendriticcells with a tumor lysate. Notably, the methods disclosed herein utilizea gentle cell lysis protocol that preserves the integrity of the tumorantigen. This gentle lysis may be achieved by exposing the tumor orcancer cells to a calcium or sodium hypochlorite solution for no morethan about 30-60 minutes. Similarly, any tumor cells used to primedendritic cells are disassociated gently, for instance, by a MiltenyiGentleMACS system, or the like.

Primed dendritic cells prepared by the methods disclosed herein may beadministered to the subject along with an agent that will boost thesubject's immune system. For example, the primed dendritic cells may beadministered to the subject after infecting the subject with a virus. Byway of example, the methods and examples disclosed herein use a Denguevirus, particularly Dengue virus serotype 2 strain #1710, which isrelatively safe (e.g., no known occurrence of lethality or seriousadverse events). This virus provides for the activation of a suppressedimmune system (e.g., by producing a T_(H)1 polarity shift), andimproving targeting specificity, thereby providing higher efficacy andsafety relative to current cellular therapies. The combination of primeddendritic cells with a viral infection provides for an effectivetreatment with minimal administration, possibly as few as one time,which avoids the challenge of subject adherence to therapy. The primeddendritic cells may be autologous, meaning derived from a subject's owncells, or allogenic, derived from another subject with a similar tissuetype.

DEFINITIONS

Throughout this disclosure, various embodiments are presented in a rangeformat. It should be understood that the description in range format ismerely for convenience and brevity and should not be construed as aninflexible limitation on the scope of any embodiments. Accordingly, thedescription of a range should be considered to have specificallydisclosed all the possible subranges as well as individual numericalvalues within that range to the tenth of the unit of the lower limitunless the context clearly dictates otherwise. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual valueswithin that range, for example, 1.1, 2, 2.3, 5, and 5.9. This appliesregardless of the breadth of the range. The upper and lower limits ofthese intervening ranges may independently be included in the smallerranges, and are also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the invention, unless thecontext clearly dictates otherwise.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of any embodiment.As used herein, the singular forms “a,” “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof. As used herein, the term “and/or”includes any and all combinations of one or more of the associatedlisted items.

Unless specifically stated or obvious from context, as used herein, theterm “about” in reference to a number or range of numbers is understoodto mean the stated number and numbers+/−10% thereof, or 10% below thelower listed limit and 10% above the higher listed limit for the valueslisted for a range.

The term “subject” as used herein includes to mammals. Mammals includerats, mice, non-human primates, and primates, including humans.

Methods of Isolating and Priming Dendritic Cells (DC)

Provided herein are methods for priming DCs and administering the primedDCs to a subject in need thereof, wherein the DC induce a response fromcytotoxic T lymphocytes (CTL) resulting in cytotoxicity of target cells.The DCs may comprise allogeneic dendritic cells or autologous dendriticcells. In some instances, the methods described herein compriseadministering allogeneic primed dendritic cells to a subject. In someinstances, the methods described herein comprise administeringautologous primed dendritic cells to a subject. The methods disclosedherein comprising administering primed DCs to the subject may bereferred to herein as “dendritic cell vaccination.”

In some instances, methods described herein comprise obtaining dendriticcells from CD34⁺ progenitor cells in the bone marrow. In some instances,methods described herein comprise obtaining dendritic cells from CD1⁺CD14⁺ immature monocytes in the peripheral blood. In some instances,obtaining the dendritic cells comprises leukapheresis. In someinstances, leukapheresis comprises withdrawing a unit of blood from thesubject or a donor, separating a series of blood-components: red cells,platelets, and most of the plasma factors, which are returned to thesubject, with the white blood cells remaining. In some instances,methods described herein comprise assessing the white blood cells forsterility, shipping or storing them cold (4° C.), and or processing theDCs from the apheresis product.

Methods of DCs production disclosed herein may comprise separatingmonocytes in the unit of blood from other white cells, including, butnot limited to, T cells, B cells, NK cells, Eosinophils and Basophils.This may be accomplished with immuno-magnetic selection or by adherenceproperties. Immuno-magnetic selection involves contacting white bloodcells from the unit of blood with a sterile plastic column with plasticbeads coated with antibodies for immune cells, such as, by way ofnon-limiting example, CD surface proteins: (CD4, CD8, CD56, etc.).Unwanted (non-monocyte) cells will adhere to the beads, leaving themonocytes to pass through and be collected. In positive selection,magnetic beads may be coated with antibodies for CD1 and/or CD14 tocapture monocytes, a magnet is placed against the column, and unwantedcells are flushed out of the column with a buffered saline solution orcell-viable media. The monocytes are then washed off the beads andcollected in a following step. In adherence selection, the properties ofmonocytes to stick to certain surfaces are used to separate them byrunning the apheresis product down a slanted column.

Provided herein are methods for cell collection which may comprisecollecting only a few thousand monocytes from the unit of blood.Effective immunotherapy generally requires DC doses in the range of 50million. Thus, methods disclosed herein may comprise expandingmonocytes, as well as any precursors thereof, and any cellsdifferentiated therefrom (e.g., DCs). Expanding cells may comprisecontacting cells with factors such as growth factors, colony-stimulationfactors, cytokines, or any other proliferation or growth inducingfactors, and combinations thereof. By way of non-limiting example, therecombinant human growth factors rhuInterleukin-4 (IL-4), andrhuGranulocyte-Macrophage-Colony-Stimulation Factor (GM-CSF), may beused to accomplish the expansion of DC numbers. In addition, IL-4 andGM-CSF may be required to develop mature DCs from monocytes, which havepoor antigen-uptake and CTL-stimulating ability, compared to mature DCs.Thus, IL-4 and GM-CSF may expand the number and the development ofmature-DC markers. DC markers may include, but are not limited to CD11,CD80, and CD83, as well as increased expression of both Class I (forpresentation of short peptides to CD8⁺ cells), and Class II (forpresentation of longer peptides to CD4⁺ Helper-Inducer T lymphocytes)MHC complexes. Expanding cells may produce mature D DCs C in the tens ofmillions within about 2 days. Expanding cells may produce mature DCs inthe tens of millions within about 3 days. Expanding cells may producemature DCs in the tens of millions within about 4 days. Expanding cellsmay produce mature DCs in the tens of millions within about 5 days.Expanding cells may produce mature DCs in the tens of millions withinabout one week.

In some instances, methods described herein comprise contacting orpulsing DCs with peptides/antigens, tumor cells, tumor supporting cells,tumor cell lysate and/or tumor supporting cell lysate. The term“pulsing,” as used herein, generally refers to contacting the cells morethan once at one or more intervals, and may be used interchangeably withcontacting, unless specified otherwise. In some instances, the methodscomprise contacting or pulsing DCs with a peptide that binds MHC Class Imolecules (“MHC Class I peptide”). In some instances, methods describedherein comprise contacting or pulsing DC with a peptide that binds MHCClass II molecules (“MHC Class II peptides”). In some instances, methodsdescribed herein comprise contacting or pulsing DC with MHC Class Ipeptides and MHC Class II peptides. In some instances, the contacting orpulsing makes the DCs competent to prime CTL and target CTL to tumors.In some instances, methods described here comprise contacting or pulsingDC with manufactured/synthetic Class I and/or Class II peptides. In someinstances, the Class I and/or class II peptides are manufactured, thenadded to the DC medium, optionally in in microgram quantities or less.In some instances, methods described herein include Class II peptidesfor a sustained immune response. In some instances, methods describedherein comprise DNA or RNA sequencing of the peptide (i.e. tumorantigen) and/or using electroporation to insert the DNA or RNA into theDCs to trigger antigen processing. In some instances, methods describedherein do not require HLA matching of DCs. In some instances, thepeptide or portion thereof is represented by an amino acid sequenceselected from EGSRNQDWL (SEQ ID NO:1), (TAYRYHLL) (SEQ ID NO: 2), orcombinations thereof.

Class I peptides may by manufactured, then added to the DC medium inmicrogram quantities. However, this technique is costly, because thepeptides must be matched to the subject's HLA type, and if the tumorcell does not present that antigen, it can evade detection and lysis.The lack of Class II peptides to activate CD4⁺ help leads to rapiddecline of immune response power. Other methods may comprise RNAsequencing of common tumor antigens, then using electroporation toinsert the RNA into the DCs to trigger antigen processing. This methoddoes not require HLA matching, and includes Class II peptides for asustained immune response. However, RNA sequencing may be technicallycomplex, and may only present a limited number of antigens of thousandsof potential gene products. For these reasons, autologous whole-tumorcells or their lysate have the advantages of low cost, readyavailability by biopsy (1-2 gm sufficient), and contain the full arrayof potential antigens for a broad and deep immune response.

Methods for dendritic cell priming described herein may compriseobtaining whole tumor cells and/or lysates thereof. Tumor cells may bekilled by radiation or other means and preparing lysate by variousmethods. In some instances, lysing the tumor cells does not comprisetrypsin enzyme digestion and freeze-thaw cycles, which are simple andfast, but can damage the delicate peptides within. The methods disclosedherein may employ an automated cell processor (e.g. the MiltenyiGentleMACS system), which allows the sample to be manually minced,suspended in PBS solution, then a pre-selected tissue-specificsoftware-controlled rotor system separates the tumor cells. Thesingle-cell suspension may be membrane-lysed with minimal damage totumor peptides.

Methods for dendritic cell priming described herein may comprisecontacting the dendritic cells with autologous tumor cells or lysatesthereof. In some instances, methods described herein comprise contactingthe dendritic cells with autologous whole-tumor cells (e.g. tumor cellsand tumor supporting cells) or lysates thereof which contain the fullarray of potential antigens for a broad and deep immune response.Methods for dendritic cell priming described herein may comprisecontacting the dendritic cells with tumor cell lysate comprisingapoptotic or necrotic bodies. In further instances, the tumor celllysate comprises tumor antigens from the microenvironment surroundingthe tumor cells, such as extracellular matrix proteins.

Methods for dendritic cell priming described herein may comprisecontacting the DCs with an augmenting agent that will augment thepriming, proliferation or viability of the DCs. By way of non-limitingexample, the augmenting agent may be selected from lymphokines,monokines, cytokines, growth factors, cells, cell fragments,(non-protein) small molecules, antibodies, antibody fragments, nucleicacids, and combinations thereof.

Methods for preparing cells and antigens for DC priming may compriserendering the target cells (e.g., cancer cells) incapable of celldivision. For example, the methods may comprise treating cells withmytomycin C or radiation to render cells incapable of cell division.These may include cells that are added as augmenting agents or cellsused to pulse DCs (e.g., tumor cells).

In some instances, methods described herein comprise pulsing the DCsfrom about 1 hour to about 24 hours. In some instances, methodsdescribed herein comprise pulsing the DCs from about 12 hours to about48 hours. In some instances, methods described herein comprise pulsingthe DCs from about 8 hours to about 24 hours. In some instances, methodsdescribed herein comprise pulsing the DCs for about 18 hours. Pulsingmay comprise contacting the DCs at least once with thepeptides/antigens, tumor cells, tumor supporting cells, tumor celllysate and/or tumor supporting cell lysate. Pulsing may comprisecontacting the DCs at least twice with the peptides/antigens, tumorcells, tumor supporting cells, tumor cell lysate and/or tumor supportingcell lysate. Pulsing may comprise contacting the DCs at least threetimes with the peptides/antigens, tumor cells, tumor supporting cells,tumor cell lysate and/or tumor supporting cell lysate. Pulsing maycomprise contacting the DCs less than two times, less than three times,less than four times, less than five times, or less than 10 times withthe peptides/antigens, tumor cells, tumor supporting cells, tumor celllysate and/or tumor supporting cell lysate. Pulsing may comprise addingthe peptides/antigens, tumor cells, tumor supporting cells, tumor celllysate and/or tumor supporting cell lysate to the DC more than once,such that the peptides/antigens, tumor cells, tumor supporting cells,tumor cell lysate and/or tumor supporting cell lysate accumulates in theDC culture media. Pulsing may comprise washing the cells or removing theDC culture media between one or more pulses.

Methods described herein may comprise contacting DC with a maturingagent to enhance, complete or finalize the maturation of the DC. IN someembodiments, the maturing agent also acts as a “danger signal.” Withoutthis danger signal, the tumor antigen may induce T^(reg) production oractivity, which will ultimately lower CTL activity. In some embodiments,the maturing agent/danger signal is an inflammatory signal. Theinflammatory signal may also be referred to as an inflammatory mediator.Inflammatory mediators may include cytokines, as well as other factors(e.g., chemokines, adhesion molecules, etc.), that may not be classifiedby those in the art as cytokines, but affect inflammation eitherdirectly or indirectly, In some embodiments, the inflammatory mediatoris selected from a chemokine, a cytokine, a pathogen, a non-peptidicsmall molecule, a compound, an antibody, a peptide, fragments thereof,portions thereof, and combinations thereof. In some embodiments, theinflammatory signal is a modulator of a pattern recognition receptor(PRR) or pathway thereof.

In some embodiments, inflammatory signals are selected from aninterferon, a toll-like receptor signaling modulator, and combinationsthereof. By way of non-limiting example, the interferon may beinterferon-gamma. In some embodiments, the inflammatory signal is atoll-like receptor signaling pathway modulator.

In some embodiments, the inflammatory signal is a toll-like receptor(TLR) signaling pathway regulator. By way of non-limiting example, thetoll-like receptor signaling pathway regulator may be lipopolysaccharide(LPS), a polysaccharide from bacterial cell walls.

The toll-like receptor signaling pathway regulator may be selected froma toll-like receptor signaling pathway regulator that regulates TLR1,TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 and TLR 10. The toll-likereceptor signaling pathway regulator may be a ligand, a binding protein,an antibody, an agonist or an antagonist, of a TLR. The toll-likereceptor signaling pathway regulator may be selected from a peptide, aprotein, a cell fragment, a cell-wall component, a lipoprotein, apeptidoglycan, a polysaccharide, a monosaccharide, and a small moleculecompound. The toll-like receptor signaling pathway regulator may be aportion of an animal cell, a plant cell, a bacterial cell, a yeast cell,a fungal cell, and combinations thereof. The toll-like receptorsignaling pathway regulator may be a TLR2 signaling pathway regulator.By way of non-limiting example, the TLR2 signaling pathway regulator maybe lipoteichoic acid, MALP-2, MALP-4, OspA, Porin, LcrV, lipomannan, GPIanchor, lysophosphatidylserine, lipophosphoglycan,glycophosphatidylinositol, zymosan, hsp60, and hemagllutinin. Thetoll-like receptor signaling pathway regulator may be a TLR4 signalingpathway regulator. By way of non-limiting example, the TLR4 signalingpathway regulator may be buprenorphine, carbamazepine, ethanol,fentanyl, levorphanol, LPS, methadone, morphine, oxcarbazepine,oxycodone, pethidine, and glucuronoxylomannan. The toll-like receptorsignaling pathway regulator may be a TLR7 signaling pathway regulator.By way of non-limiting example, the TLR7 signaling pathway regulator maybe a single stranded RNA or an imidazoquinoline compound. The toll-likereceptor signaling pathway regulator may be a TLR8 signaling pathwayregulator. By way of non-limiting example, the TLR8 signaling pathwayregulator may be a single stranded RNA, a G-rich oligonucleotide or animidazoquinoline compound. The imidazolquinoline compound may be R848.

After exposure to the inflammatory signal, the DC may up-regulate theirCD80/CD83⁺ activation markers, increase production of IL-12p70 to inducea Type 1 CTL response, and become resistant to further antigen uptakeand processing.

Methods for producing primed dendritic cells described herein maycomprise contacting primed dendritic cells with interferon gamma. Insome embodiments, the methods comprise culturing the primed dendriticcells in a culture media with a concentration of interferon gammaselected from about 100 U/mL to about 10,000 U/mL, about 500 U/mL toabout 5000 U/mL, and about 500 U/mL to about 2,000 U/mL. In someembodiments, the methods comprise culturing the primed dendritic cellsin a culture media with a concentration of interferon gamma of about 500U/mL. In some embodiments, the methods comprise culturing the primeddendritic cells in a culture media with a concentration of interferongamma of about 1000 U/mL. In some embodiments, the methods compriseculturing the primed dendritic cells in a culture media with aconcentration of interferon gamma of about 2000 U/mL.

Methods for producing primed dendritic cells described herein maycomprise contacting primed dendritic cells with TLR8 agonist R848. Insome embodiments, the methods comprise culturing the primed dendriticcells in a culture media with a concentration of R848 selected fromabout 0.1 μg/mL to about 50 μg/mL, about 1 μg/mL to about 20 μg/mL, andabout 1 μg/mL to about 10 μg/mL. In some embodiments, the methodscomprise culturing the primed dendritic cells in a culture media with aconcentration of R848 of about 1 μg/mL. In some embodiments, the methodscomprise culturing the primed dendritic cells in a culture media with aconcentration of R848 of about 5 μg/mL. In some embodiments, the methodscomprise culturing the primed dendritic cells in a culture media with aconcentration of R848 of about 10 μg/mL.

Methods for producing primed dendritic cells described herein maycomprise contacting primed dendritic cells with lipopolysaccharide. Insome embodiments, the methods comprise culturing the primed dendriticcells in a culture media with a concentration of lipopolysaccharideselected from about 1 ng/mL to about 100 ng/mL, about 1 ng/mL to about50 ng/mL, and about 1 ng/mL to about 25 ng/mL. In some embodiments, themethods comprise culturing the primed dendritic cells in a culture mediawith a concentration of lipopolysaccharide of about 5 ng/mL. In someembodiments, the methods comprise culturing the primed dendritic cellsin a culture media with a concentration of lipopolysaccharide of about10 ng/mL. In some embodiments, the methods comprise culturing the primeddendritic cells in a culture media with a concentration oflipopolysaccharide of about 15 ng/mL.

Methods described herein may comprise sterility, specificity, andviability assessment of the DCs. The testing may occur before shippingor storing the DCs. The testing may occur after shipping or storing theDCs. The methods may comprise measuring expression level of IL-12p70 inDCs, either at the RNA or protein level. IL-12p70 is an independentpredictor of clinical response, tested across numerous trials in thelast two decades, some with about 40% response rates. The expressionlevel of IL-12p70 in primed DCs produced by the methods disclosed hereinmay be at least about two times greater than primed DCsproduced/stored/shipped by traditional methods. The expression level ofIL-12p70 in primed DCs produced by the methods disclosed herein may beat least about two times greater than primed DCs produced/stored/shippedby traditional methods (“traditional primed DC”). The expression levelof IL-12p70 in primed DCs may be at least about 10%, at least about 20%,at least about 30%, at least about 40%, at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, at least about 90%,or at least about 100% greater than traditional primed DCs. Theexpression level of IL-12p70 in primed DCs may be at least about threetimes greater than traditional primed DCs. The expression level ofIL-12p70 in primed DCs may be at least about four times greater thantraditional primed DCs. The expression level of IL-12p70 in primed DCsproduced by the methods disclosed herein may be about two to abouttwenty times greater than traditional primed DCs.

Provided herein are dendritic cells that produce more than 6 ng/mL ofIL-12p70. Also provided herein are dendritic cells that produce morethan 10 ng/mL of IL-12p70. In some instances, DCs produced by methodsdescribed herein produce at least about 10 ng/mL, at least about 12ng/mL, at least about 14 ng/mL, at least about 16 ng/mL, at least about18 ng/mL, at least about 20 ng/mL, at least about 22 ng/mL, or at leastabout 24 ng/mL. In some instances, DCs produced by methods describedherein produce about 10 ng/mL to about 30 ng/mL. In some instances, DCsproduced by methods described herein produce from about 10 ng/mL toabout 25 ng/mL. In some instances, DCs produced by methods describedherein produce from about 15 ng/mL to at least about 23 ng/mL. In someinstances, DCs produced by methods described herein produce from about6.5 ng/mL to at least about 23 ng/mL.

CTL Response

Methods for producing DCs described herein may comprise testing theability of the DCs to induce a CTL response. Measuring the level of theCTL response may comprise measuring cytokines or inflammatory mediatorsin blood, serum or plasma from the subject. Measuring the level of theCTL response may comprise measuring a change in the level of a cytokineor inflammatory mediator in blood, serum or plasma from the subject.Measuring the level of the CTL response may comprise measuring theproduction of a cytokine or inflammatory mediator in vitro. Cytokinesand inflammatory mediators may include interleukins, migrationinhibitory proteins, monocyte chemotactic proteins, monocytechemoattractant proteins, interferons, tumor necrosis factors, colonystimulating factors (CSFs), macrophage inflammatory proteins, monokines,chemokines, chemokine ligands (CCLs), and C-X-C motif chemokines (CXCL),and receptors thereof. Cytokines and inflammatory mediators include, butare certainly not limited to, interleukin 1 beta (IL-1b), interleukin 2(IL-2), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 7(IL-7), interleukin 8 (IL-5), interleukin 10 (IL-10), interleukin 13(IL-13), interleukin 6 (IL-6), interleukin 12 (IL-12), interleukin 15(IL-15), interleukin 17 (IL-17), Rantes, Eotaxin, macrophageinflammatory protein 1 alpha (MIP-1a), macrophage inflammatory protein 1beta (MIP-1b), granulocyte macrophage colony-stimulating factor(GM-CSF), monocyte chemoattractant protein-1 (MCP-1), interferon alpha(IFNa), interferon gamma (IFNg), interleukin 1 receptor alpha (IL-1Ra),interleukin 2 receptor (IL-2R), tumor necrosis factor alpha (TNFa),interferon gamma induced protein (IP-10), and monokine induced by gammainterferon (MIG). CTL response may be measured by expression of tumorresponse genes (MxA, etc.), enabling high cancer killing (turning “cold”tumors “hot”), and generating further tumor shrinkage in non-responderor low responders.

Hard Surface

Methods for DC preparation described herein may comprise culturing DCson a hard surface. The term, “hard surface,” as used herein, generallyrefers to a standard plastic tissue culture plate or flask (e.g. apolystyrene plate). The methods disclosed herein comprise culturing DCson a hard surface to which the DCs can adhere. In some embodiments, thehard surface is coated with a protein, peptide, extracellular matrixmolecule, polymer, or combinations thereof. In some embodiments, thehard surface is not coated (e.g., the DCs adhere directly to the hardplastic surface). The hard surface is contrasted to a soft tissueculture bag, also known as cell differentiation bags. Soft tissueculture bags may be bags comprising polymers or chemicals (e.g.phthalates) that reduce the DC's Type 1 response capability. Soft tissueculture bags may be bags comprising polymers or chemicals that evoke aneutral Type 0 response from the DCs, rendering the DCs functionallyinert. Soft tissue culture bags may be bags comprising a polymerselected from polyethylene, fluorinated ethylene propylene (FEP),hexafluoropropylene, tetrafluoroethylene, polytetrafluoroethylene, andco-polymers thereof, and combinations thereof.

Methods for DC preparation described herein may comprise transferringthe DCs to a storage unit. The storage unit may also be a shipping unit.The storage unit may be selected from a flexible or soft container orsurface (e.g., a bag) or a hard container or surface (e.g., a flask orplate). The storage unit may comprise a hard plastic surface. Thestorage unit may consist essentially of a hard plastic surface. Thestorage unit may consist of a hard plastic surface. The storage unit maycomprise a non-plastic surface (e.g., glass). The storage unit mayconsist essentially of a non-plastic surface. The storage unit mayconsist of a non-plastic surface. The storage unit may be free of anypolymers that would be taken up by, and/or induce a response in, cellsstored within the storage unit. The storage unit may be free oressentially free of polymers that induce a neutral or Type 0 response inimmature DCs. A neutral response may be characterized by low expressionof IL-12p70. The storage unit may be essentially free of any polymersthat would be taken up by, and/or induce a response in, cells storedwithin the storage unit. Essentially free may mean that the storage unitis at least 90%, at least 95%, at least 98%, or at least 99% free of anypolymers that would be taken up by, and/or induce a response in, cellsstored within the storage unit. Essentially free may mean that thestorage unit is at least 99.5%, at least 99.6%, at least 99.7%, at least99.8%, or at least 99.9% free of any polymers that would be taken up by,and/or induce a response in, cells stored within the storage unit.

Provided herein are storage units for storing DCs produced by methodsdescribed herein, wherein the storage units comprise an inner surface,wherein the inner surface is the surface of the storage unit that is incontact with cells stored therein. The inner surface may consist of ahard plastic surface. The inner surface may be glass. The inner surfacemay be absent of any polymers that would be taken up by, and/or induce aresponse in, cells stored within the storage unit. The inner surface maybe constructed of polymers that are not taken up by immature DC or anycells stored within the storage unit. The inner surface may be free ofany polymers that would be taken up by, and/or induce a response in,cells stored within the storage unit. The inner surface may beessentially free of any polymers that would be taken up by, and/orinduce a response in, cells stored within the storage unit. The innersurface may be at least 90%, at least 95%, at least 98%, or at least 99%free of any polymers that would be taken up by, and/or induce a responsein, cells stored within the storage unit following addition of cells andstorage media. The inner surface may be at least 99.5%, at least 99.6%,at least 99.7%, at least 99.8%, or at least 99.9% free of any polymersthat would be taken up by, and/or induce a response in, cells storedwithin the storage unit following addition of cells and storage media.The inner surface may be free or essentially free of polymers thatinduce a neutral or Type 0 response in immature DCs. A neutral responsemay be characterized by low expression of IL-12p70.

Provided herein are storage units for storing DCs produced by methodsdescribed herein, wherein the storage units are suitable for freezing at−70° C. in liquid N₂, storage up to 1 year, and shipping to the clinicfor use. The methods may comprise storing and/or shipping mature DCs,immature DCs, monocytes or blood in a storage unit. The methods maycomprise shipping cells cool overnight. The methods may comprise thawingor warming cells to 37° C. (e.g., in a warm-water bath).

Methods of Isolating and Lysing Tumor Cells

Provided herein are methods for treating a subject, comprisingadministering the DCs disclosed herein to target tumor cells. In someinstances, DCs are primed with tumor cells from a subject. In someinstances, the tumor cells are isolated cells from a tumormicroenvironment of the subject, referred to herein as tumor supportingcells. In some instances, dendritic cells are exposed to/pulsed withtumor cells, tumor supporting cells and/or peptides thereof, such thatthe dendritic cells will target tumor cells and/or tumor supportingcells that support tumor growth and metastasis (e.g. endothelial cells,vascular cells, immune cells, etc.). In some instances,peptides/antigens from tumor cells and tumor supporting cells inducedendritic cells or cytotoxic lymphocytes with receptors forpeptides/antigens on both tumor cells and tumor supporting cells,resulting in targeting of the dendritic cells or cytotoxic lymphocytesto the tumor microenvironment rather than only the tumor cells. In someinstances, tumor cells and/or tumor supporting cells are obtained from abiopsy of tumor tissue. In some instances, the biopsy comprises cellsselected from tumor cells, adipocytes, fibroblasts, endothelial cells,infiltrating immune cells, and combinations thereof. In someembodiments, the methods comprise expanding tumor cells in order to havea sufficient number of tumor cells, tumor cell lysates or tumor cellantigens to effectively and optimally prime/pulse the DC. Expanding maycomprise proliferating of the tumor cells in vitro.

Provided herein are methods for activating DCs disclosed herein totarget tumor cells, wherein the DCs are activated with lysed tumor cellsand/or tumor supporting cells and surrounding extracellular matrix. Insome instances, lysing comprises contacting the tumor cells and/or tumorsupporting cells with an NH₄Cl enzyme solution to eliminate red bloodcells. In some instances, the lysing comprises contacting the tumorcells and/or tumor supporting cells with hypochlorous acid solution toinduce immunogenic cell death. In some instances, the cells are lysedgently enough to not destroy peptides. In some instances, the cells arelysed to produce apoptotic or necrotic bodies. In some instances, themethods comprise lysing the tumor cells and/or tumor supporting cellswith an enzymatic solution. In some instances, the methods compriselysing the tumor cells and/or tumor supporting cells with aperoxide-free solution or a low peroxide-containing solution.

Provided herein are methods for activating DCs disclosed hereincomprising lysing the tumor cells with a hypochlorite solution (HOCL).In some instances, the hypochlorite solution comprises sodium chlorite.In some instances, the hypochlorite solution comprises calcium chlorite.In some instances, the concentration of the hypochlorite in a media inwhich the tumor cells are suspended is about 10 μM, about 20 μM, about30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM,about 90 μM, or about 100 μM.

Provided herein are methods for methods activating DCs comprise lysingthe tumor cells and/or tumor supporting cells with a detergent solutionprior to contact with the DCs. In some instances, the detergent isselected from, but is not limited to, Triton X-100, Triton X-114, NP-40,Brij-35, Brij-58, Tween 20, Tween 80, octyl glucoside, octylthioglucoside, SDS, CHAPS, and CHAPSO. In some instances, the detergentsolution is purified of peroxides, and other impurities. In someinstances, the detergent is about 0.1% to about 10% v/v of the detergentsolution. In some instances, the detergent is about 0.1% to about 5% v/vof the detergent solution. In some instances, the detergent is about0.5% to about 5% v/v of the detergent solution. In some instances, thedetergent is about 1% to about 10% v/v of the detergent solution. Insome instances, the detergent is about 1% to about 5% v/v of thedetergent solution. In some instances, the methods comprise lysing cellswithout shaking, vortexing, freezing, thawing, shear pressure,sonicating and/or heating the cells.

In some instances, methods for cell lysis described herein furthercomprise stopping or neutralizing the lysing. For example, cells may bewashed with a buffered saline solution (phospho-buffered saline solutionor Hank's balanced salt solution) to neutralize the lysing.

Combination Therapy

Provided herein are combination therapies comprising therapeutic agentsdisclosed herein with other types of therapies in order to achieve anoptimal result. For example, in some instances, combination approachesto cancer immunotherapy may be more successful than single-axis attackswhich tumors can mutate to avoid. In some embodiments, the therapy is acancer therapy. Cancer therapies include, but are not limited to,chemotherapy, radiation, small molecule inhibitors, and monoclonalantibodies.

Provided herein are compositions and methods wherein dendritic cellvaccination is combined with an adjuvant effect of a virus to overcometumor immune evasion mechanisms and deplete tumor cells. A schematicrepresentation of the combination therapies disclosed herein is depictedin FIG. 1. Methods described here may be used to treat a subject 101 forcancer by obtaining 101 dendritic cells 102 and tumor cells 104 from thesubject, exposing the dendritic cells to the tumor cells or tumor celllysate 105, also referred to as “pulsing” the dendritic cells, to primed(or “activated”) the dendritic cells, delivering 107 the resultingprimed and tumor-targeting dendritic cells to the subject after thesubject has had his/her immune system stimulated with the virus 108(see, e.g., FIG. 1). Optionally, a tumor antigen that is not from thesubject can be used for pulsing the dendritic cells.

Tumor immune evasion mechanisms are responsible for the lack of efficacyseen with most immunotherapy platforms. Compositions and methodsdescribed herein provide for a multi-pronged approach, combiningphysiological (hyperthermic reduction of tumor perfusion), immunological(activation of effector cells of the adaptive and innate immune system),and apoptosis-inducing pathways (sTRAIL) to destroy tumor cells. Using avirus, like Dengue virus (DV), as an adjuvant to activate many pathwaysworking in synergy may support the eradication of mutated tumor cells,improving the clinical efficacy of the cancer immunotherapy. Methodsdescribed herein provide cancer immunotherapies based on multiplemechanisms of action in concert and result in a decline in the abilityof the tumor cells to employ resistance methods compared to delivery ofany single method along.

Provided herein are methods for treating a subject having a disease orcondition, comprising: obtaining dendritic cells (DCs); incubating theDCs with at least one tumor cell antigen; administering a virus to thesubject; and administering the DCs to the subject. In some instances,the dendritic cells are autologous dendritic cells. In some instances,the dendritic cells are allogeneic dendritic cells. In some instances,incubating the DCs with at least one tumor antigen comprises incubatingthe DCs with a tumor cell. In some instances, incubating the DCs with atleast one tumor antigen comprises incubating the DCs with a tumor celllysate. In some instances, incubating the DCs with at least one tumorantigen comprises incubating the DC with a peptide expressed by a tumorcell. In some embodiments, the condition or disease is cancer. In someembodiments, the virus is an Arbovirus. In some embodiments, the virusis a Dengue virus.

Disclosed herein are methods for treating cancer in a subject in needthereof, comprising: obtaining dendritic cells (DCs); incubating the DCswith at least one tumor cell antigen; administering a Dengue Virus Type2 serotype strain to the subject; and administering the DCs to thesubject. In some instances, the Dengue Virus Type 2 serotype strain isDENV-2 #1710. In some instances, the dendritic cells are autologousdendritic cells. In some instances, the dendritic cells are allogeneicdendritic cells. In some instances, incubating the DCs with at least onetumor antigen comprises incubating the DCs with a tumor cell. In someinstances, incubating the DCs with at least one tumor antigen comprisesincubating the DCs with a tumor cell lysate.

Administering the virus may increase cancer cell death or cancer celllysis beyond that induced by DCs alone. Cancer cell death may beincreased by at least about 10% to 25%, at least about 10% to about 50%,at least about 20% to about 100%, at least about at least about 20% toabout 200%. Administering the virus may reduce the size of tumor lesionsbeyond that reduced by DCs alone. Tumor lesion size may decrease by atleast about 10% to about 50%, at least about 10% to about 30%, at leastabout 15% to about 80%. Administering the virus may reduce the number ofmyeloid-derived suppressor cells (MDSC) in the tumor microenvironmentbeyond that decreased by DCs alone. Administering the virus may reducethe number of myeloid-derived suppressor cells (MDSC) in the tumormicroenvironment by at least about 10% to about 65%, at least about 10%to about 85%, at least about 10% to about 100%, or at least about 10% toabout 200%.

Dengue Viruses

Provided herein are methods for combination therapy comprisingadministering a Dengue virus (DV) and activated DCs disclosed herein totarget tumor cells, wherein the DV is administered to a subject. As usedherein, the term “Dengue virus” includes any serotype of Dengue virusserotypes 1, 2, 3, 4, or 5. The term Dengue virus may also encompassgenetically modified DV, in vitro mutated DV, and combinations of DV orproteins/peptides thereof. The DV may be alive, dead, recombinant or aprotein/peptide thereof.

In primary infections, the death rate from DV is very low (1 in 61,000per Manson's Tropical Diseases). The virus infects, but does not killAPC of the monocyte-macrophage and Dendritic Cell lineage. Theseinfected APC then begin a cytokine cascade of the pro-inflammatory(TNF-alpha and IL-1 beta), and T_(H)1 (IL-2, IL-7, IL-12, IL-15, andIL-21) types. These cytokines may result in strong activation of boththe adaptive (CTL) and innate (NK) immune systems. After a 3-5 dayincubation period, the fever rises to 39.5-40.5° C., and remainselevated for 4-5 days. The subject experiences intense headache, jointpain, malaise, and sensitivity to light. A rash covering the chest,back, and sometimes legs and arms, may develop by day 3 of fever.Clinically, dengue infections result in lowered platelet counts leadingto hemorrhage, which ranges from minor to life-threatening in case ofshock syndrome. With proper supportive care based on judicious fluidmanagement, recovery is complete in 99% of cases.

Dengue viruses are Arboviruses, and are transmitted exclusively bymosquitoes of the Aedes aegypti and albopictus species. The virus has acomplex life cycle involving an unidentified forest-dwelling mammalianreservoir (possibly primates), and human hosts. The female mosquitotakes a blood meal from an infected person, the virus replicates to ahigh infectious titer (10^5/ml), in gut epithelial cells, then istransmitted to another person when the mosquito withdraws its styletusing back pressure after another blood meal. Dengue epidemics infect 50million persons annually, with several thousand deaths, usually childrenwith inadequate treatment of secondary infection-related shock.

The Dengue virus genome encodes structural proteins, capsid protein C,membrane protein M, envelope protein E, and nonstructural proteins, NS1,NS2a, NS2b, NS3, NS4a, NS4b and NS5. In some instances, the Dengue virusis a live strain of the Dengue virus. In some instances, the Denguevirus is an attenuated strain of the Dengue virus. In some instances,the Dengue virus is a weakened strain of the Dengue virus. In someinstances, the Dengue virus is selected the following serotypes ofdengue virus: DENV-1, DENV-2, DENV-3, DENV-4, and DENV-5, andcombinations thereof.

Dengue Viruses are positive-strand RNA viruses of the Togavirus Family,sub-family Flaviviridae, (Group B). The virus has an icoashederalgeometry and is approximately 40-45 nanometers in diameter. The 11,000base genome codes for a nucleocapsid (NC), protein, a prM membranefusion protein, an envelope glycoprotein (E), and 5 non-structuralproteins NS1-NS5. The NC protein forms the viral core, with the envelopespikes attached via the prM complex. The E glycoprotein is the maintarget of neutralizing antibodies, and the NS-3 and NS-4 proteins makeup the main targets for CD4+ and CD8+CTL.

The dengue viruses make up five distinct serotypes, DENV-1 throughDENV-5. The serotypes 2 and 4 are cross-neutralizing for IgG, and types1 and 3 are also cross-neutralizing. Immunity is not complete, however,and dengue is unique among viral infections in that a subsequentinfection by a non-cross-neutralizing serotype carries an increased riskof mortality due to shock syndrome from immune hyper-activation.

Provided herein are compositions and method using such compositions,wherein the composition comprises Dengue virus serotype 1, 2, 3, 4, or5. In some instances, the DV is serotype 2. In some instances the DVserotype 2 is DENV-2 strain #1710. DENV-2 strain #1710 may beadvantageous over other DV strains because it is milder at infectingsubjects, and are therefore safer. Other more virulent strains may havea stronger anti-tumor effect, but they may not be suitable due to safetyconcerns. A more virulent strain, by way of non-limiting example, isDENV-2 strain #1584. The DENV-2 strain #1710 is from a sample taken fromPuerto Rico in 1985 and characterized as type A from a restriction sitespecific RT-PCR analysis using 4 primers (see Table 1) specific to theenvelope gene region. See Harris et al., Virology 253, 86-95 (1999).Restriction site specific RT-PCR with these primers producesamplification products of 582 base pairs, 754 base pairs, and possibly676 base pairs. The DENV-2 strain #1710 is recorded in a CDC database asentry number 555. See Harris (1999). The DENV-2 strain #1710 wasisolated during a Puerto Rican epidemic. This outbreak had 9,540suspected cases of DV, with one suspected, but no confirmed deaths dueto the virus, which indicates the toxicity of DEN-2 strain #1710 is verylow and therefore suitable for the methods disclosed herein.

TABLE 1 Sequence and Position of Primers to Amplify DENV-2 #1710 virusGenome Primer Sequence Position Strand RSS1 5′-GGATCCCAAGAAGGGGCCAT-3′1696-1715 + (SEQ ID NO: 3) RSS2 5′-GGCAGCTCCATAGATTGCT-3 2277-2259 −(SEQ ID NO: 4) RSS3 5′-GGTGTTGCTGCAGATGGAA-3′ 1524-1542 + (SEQ ID NO: 5)RSS4 5′-GTGTCACAGACAGTGAGGT-3′ 2371-2353 − (SEQ ID NO: 6)

Certain characteristics of DV and DV infection may make this virusparticularly useful for the methods disclosed herein. For example, aunique feature of DV is that primary infections result in activation ofa T_(H)1-type response of CD4+ and CD8+ helper-inducer andcytotoxic-effector CTL. By infecting, but not killing the antigenpresenting cell (APC), (e.g. dendritic cell), DV up-regulates CD80 andCD83 expression on the APC, resulting in a pro-inflammatory T_(H)1cytokine profile. Primary DV infections induce a T_(H)1 type responsewith activated CD4⁺ and CD8⁺ effector T cells as well aslymphokine-activated killer cells. This may increase the likelihood of acomplete response to a cancer immunotherapy, such as therapies utilizingthe primed dendritic cells disclosed herein.

In some cases, Dengue viruses can provide a counter attack to a tumorimmune evasion mechanism. The tumor immune evasion mechanism, by way ofnon-limiting example, may be low levels of MHC on the tumor cell toprevent CTL recognition, and the counter attack may be high Interferon-γraising MHC levels by up-regulating MHC gene expression. The tumorimmune evasion mechanism, also by way of non-limiting example, may beone or more point mutations in a tumor peptide to prevent TCR binding,and the counter attack may be stimulating lymphokine-activated killercells or cytokine-induced killer cells to target “escaped” tumor cellsexpressing aberrant peptides or MHC. In some embodiments, the tumorimmune evasion mechanism is tumor vessels lacking factors for CTLattachment and trafficking, and the counter attack is high TNF-αrestores gaps by altering PECAM-1, restores ICAM-1/VCAM-1 expression andP and E-selectins. In some embodiments, the tumor immune evasionmechanism is FasL killing of Fas⁺ CTL by triggering apoptosis, and thecounter attack is high IL-6 and/or IL-151 protects Fas⁺ CTL byup-regulating FLIP ligand. In some embodiments, the tumor immune evasionmechanism is HLA-G protects from NK Cells, and the counter attack may behigh IL-2, IL-7, IL-12, and/or IL-15 raise activation of NK. The tumorimmune evasion mechanism may be stromal barriers inhibit CTL and thecounter attack may be high IFN-γ activates macrophages to M₁. The tumorimmune evasion mechanism may be Myeloid-Derived Suppressor Cells, (MDSC)and the counter attack may be iNKT Cells decrease MDSC. The tumor immuneevasion mechanism may be CTL inactivated by TGF-β and the counter attackmay be T_(H)1 cytokines reactivate tolerant CTL. The tumor immuneevasion mechanism may be Tumor PI-9 blocks CTL killing and the counterattack may be high CD8 & ICAM-1 expression restores low-avidity CTLrecognition and lysis by stabilizing weak interactions between TCR andMHC+self-peptide. The tumor immune evasion mechanism may be T-regulatorycells block CTL and the counter attack may be high CD4^(Helper) cellsovercome CD4^(Reg) cells.

In some embodiments, the methods comprise activating or enhancing thesubject's immune response by administering Dengue virus to the subject.Activating or enhancing may comprise inducing or increasing expressionof cytokines and inflammatory mediators. Expression of genes in cells ofthe subject that may be increased by DV infection, include, but are notlimited to, IL-1 beta, IL-2, IL-7, IL-12, IL-15, IFN-alpha, IFN-gamma,TNF-alpha, TNF-beta, GM-CSF, CD8 antigen, ICOSLG, CCL3, CCL5, TRAIL,IP10, GNLY, GZMA, HLA-DRA, HLA-DP alpha1, HLA-DP beta 1, and ZAP70.

In some instances, the methods disclosed herein comprise administeringDV to the subject, wherein the administering results in the release ofTNF-α by the immune system. TNFα is an inflammatory cytokine withpleiotropic effects, including direct killing of tumor cells via TRAIL(TNF-Apoptosis-Inducing-Ligand). Increased levels of proteinscorresponding to these genes may be observed in tissues and circulatingfluids of the subject as well. Levels may be increased at least 2-fold.Levels may be increased between 2-fold and 1000-fold. Levels may beincreased between 2-fold and 100-fold. Levels may be increased between2-fold and 10-fold.

In some instances, administering DV induces high levels of soluble TRAIL(sTRAIL) from a variety of cells including γδCTL, activated M1macrophages and plasmacytoid DC (pDC). In some instances, DV activatesIFNβ, a multifunctional cytokine with a 10-fold higher affinity for thesame receptor as IFNα. IFNβ has similar antiviral properties insuppressing transcription of viral RNA, but is much more potent thanIFNα in inducing apoptosis in tumor cells. Nitric oxide and IFNβ couldact in a synergistic fashion during dengue infection. These moleculesmay work in tandem to overcome resistance to apoptosis mediated by thehigh levels of sTRAIL induced by M₁ macrophages, pDC, and δγ CTL.

Activating or enhancing the immune system of the subject may compriseinducing or increasing cell types present in the subject. These celltypes include, but are not limited to, CD8+CD44+62L-cells,CD4+CD44+CD62L^(lo) cells, HLA-DR+CD8+ cells, Tia-1 CD8+ cells, VLA-4CD8+ cells, ICAM-1 CD8+ cells, and LFA-1 CD8+ cells.

Cancer

Provided herein are methods for cancer therapy comprising administrationof therapeutic agents disclosed herein. Methods described herein alsoprovide for clearing cancer cells. In some instances, administering DVto the subject induces an immune response. In some instances, the immuneresponse is potent as compared to a common virus, such as a common coldvirus. In some instances, the immune response results in tumorregression. The methods disclosed herein may comprise developing DCscapable of inducing an immune response that results in eliminating alltumor cells in a subject's body.

DNA microarray analyses have revealed that hundreds of geneticallydistinct tumor clones may exist in a single subject with advanced tumor.There is a pattern of negative correlation between O₂ supply and geneticmutation rates. The majority of agents such as cytotoxic drugs,antibodies, and small molecules, are nearly always blood-borne, exertinga Darwinian selective pressure to tumor clones that evade therapeuticmechanisms. Clones with the lowest perfusion rates have both low drugexposure and high capacity to evade immune system detection, making themresistant to conventional therapies. Provided herein are methods forcancer cell targeting, comprising inducing fever hyperthermia byadministering DV to the subject with cancer, starving low-flow,resistant clones with mutated phenotypes, leaving more geneticallystable clones for elimination by activated lymphocytes and other arms ofthe immune system. In some instances, the methods comprise combiningfever with activation of CTL and lymphokine-activated killer cells (LAK)by administering pulsed DCs, lead to higher response rates than withconventional cancer therapies (e.g. antibody drug conjugates, kinaseinhibitors, small molecules, etc.) or CTLs alone. The immune suppressionseen in subjects with advanced cancer is a complex and dynamic process.It involves tolerance to the tumor antigens themselves, which areusually recognized as “self” by CTL. In some instances, methodsdescribed herein comprise breaking this tolerance and achieving highlevels of T_(H)1 cytokines, which DV infection induces.

Cancers targeted herein may be a recurrent and/or a refractory cancer.In some instances, the cancer is an acute cancer or a chronic cancer. Insome instances, the cancer is an accelerated refractory cancer. In someinstances, the cancer is in remission. In some instances, the cancer isa stage I, stage II, stage III, or stage IV cancer. In some instances,the cancer is a juvenile cancer or adult cancer. Examples of cancersinclude, but are not limited to, sarcomas, carcinomas, lymphomas orleukemias. In some instances, the cancer is a solid tumor or aliposarcoma.

In some instances, the cancer is a sarcoma. The sarcomas may be a cancerof the bone, cartilage, fat, muscle, blood vessels, or other connectiveor supportive tissue. In some instances, sarcomas include, but are notlimited to, bone cancer, fibrosarcoma, chondrosarcoma, Ewing's sarcoma,malignant hemangioendothelioma, malignant schwannoma, bilateralvestibular schwannoma, osteosarcoma, soft tissue sarcomas (e.g. alveolarsoft part sarcoma, angiosarcoma, cystosarcoma phylloides,dermatofibrosarcoma, desmoid tumor, epithelioid sarcoma, extraskeletalosteosarcoma, fibrosarcoma, hemangiopericytoma, hemangiosarcoma,Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma,lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma,rhabdomyosarcoma, and synovial sarcoma). The sarcoma may comprise aEwing's sarcoma.

In some instances, the cancer is a carcinoma. Carcinomas are cancersthat begin in the epithelial cells, which are cells that cover thesurface of the body, produce hormones, and make up glands. By way ofnon-limiting example, carcinomas include breast cancer, pancreaticcancer, lung cancer, colon cancer, colorectal cancer, rectal cancer,kidney cancer, bladder cancer, stomach cancer, liver cancer, ovariancancer, brain cancer, vaginal cancer, vulvar cancer, uterine cancer,oral cancer, penile cancer, testicular cancer, esophageal cancer, skincancer, cancer of the fallopian tubes, head and neck cancer,gastrointestinal stromal cancer, adenocarcinoma, cutaneous orintraocular melanoma, cancer of the anal region, cancer of the smallintestine, cancer of the endocrine system, cancer of the thyroid gland,cancer of the parathyroid gland, cancer of the adrenal gland, cancer ofthe urethra, cancer of the renal pelvis, cancer of the ureter, cancer ofthe endometrium, cancer of the cervix, cancer of the pituitary gland,neoplasms of the central nervous system (CNS), primary CNS lymphoma,brain stem glioma, and spinal axis tumors. In some instances, the canceris a skin cancer, such as a basal cell carcinoma, squamous, melanoma,nonmelanoma, or actinic (solar) keratosis. In some instances, the canceris bladder cancer.

In some instances, the cancer is a neuroendocrine cancer. In someinstances, the cancer is a pancreatic cancer. In some embodiments, thecancer is thyroid cancer. In some instances, the cancer is an epithelialcancer, breast cancer, endometrial cancer, ovarian cancer, stromalovarian cancer, or cervical cancer. In some embodiments, the cancer isprostate cancer. In some instances, the cancer is a skin cancer. In someinstances, the cancer is a neo-angiogenic skin cancer. In someinstances, the cancer is a melanoma. In some instances, the cancer is akidney cancer, a lung cancer. Exemplary lung cancers include, withoutlimitation, a small cell lung cancer or a non-small cell lung cancer. Insome instances, the cancer is a colorectal cancer, e.g., a gastriccancer or a colon cancer. In some instance, the cancer is a braincancer. In some instances, the cancer is a brain tumor. In someinstances, the cancer is a glioblastoma or an astrocytoma.

In some instances, the cancer is breast cancer. In some embodiments, thebreast cancer is a triple negative breast cancer (negative for estrogenreceptor, progesterone receptor and Her2). In some embodiments, thebreast cancer is estrogen receptor positive (ER+).

In some instances, the cancer is a lung cancer. In some instances, thelung cancer is a non-small cell lung carcinoma (NSCLC), small cell lungcarcinoma, or mesotheliomia. Examples of NSCLC include squamous cellcarcinoma, adenocarcinoma, and large cell carcinoma. In some instances,the mesothelioma is a cancerous tumor of the lining of the lung andchest cavity (pleura) or lining of the abdomen (peritoneum). In someinstances, the mesothelioma is due to asbestos exposure.

In some instances, the cancer is a central nervous system (CNS) tumor.In some instances, the CNS tumor is classified as a glioma or nonglioma.In some instances, the glioma is malignant glioma, high grade glioma,diffuse intrinsic pontine glioma. Examples of gliomas includeastrocytomas, oligodendrogliomas (or mixtures of oligodendroglioma andastocytoma elements), and ependymomas. Astrocytomas include, but are notlimited to, low-grade astrocytomas, anaplastic astrocytomas,glioblastoma multiforme, pilocytic astrocytoma, pleomorphicxanthoastrocytoma, and subependymal giant cell astrocytoma.Oligodendrogliomas include low-grade oligodendrogliomas (oroligoastrocytomas) and anaplastic oligodendriogliomas. Nongliomasinclude meningiomas, pituitary adenomas, primary CNS lymphomas, andmedulloblastomas. In some instances, the cancer is a meningioma.

In some instances, the cancer is a blood cancer. In some instances, thecancer is leukemia. In some instances, the cancer is a myeloid leukemia.In some instances, the cancer is a lymphoma. In some instances, thecancer is a non-Hodgkin's lymphoma. In some instances, the cancer isselected from myelogenous leukemia, lymphoblastic leukemia, myeloidleukemia, an acute myeloid leukemia, myelomonocytic leukemia,neutrophilic leukemia, myelodysplastic syndrome, B-cell lymphoma,burkitt lymphoma, large cell lymphoma, mixed cell lymphoma, follicularlymphoma, mantle cell lymphoma, hodgkin lymphoma, recurrent smalllymphocytic lymphoma, hairy cell leukemia, multiple myeloma, basophilicleukemia, eosinophilic leukemia, megakaryoblastic leukemia, monoblasticleukemia, monocytic leukemia, erythroleukemia, erythroid leukemia andhepatocellular carcinoma. In some instance, the cancer is ahematological malignancy. In some instance, the hematological malignancyis a B cell malignancy. In some instance, the cancer is a chroniclymphocytic leukemia. In some instance, the cancer is an acutelymphoblastic leukemia. In some instance, the cancer is a CD19-positiveBurkitt's lymphoma. In some instance, the leukemia is an acutelymphocytic leukemia, acute myelocytic leukemia, chronic lymphocyticleukemia, or chronic myelocytic leukemia. Additional types of leukemiasinclude, but are not limited to, hairy cell leukemia, chronicmyelomonocytic leukemia, and juvenile myelomonocytic leukemia.

In some instances, the lymphoma develops from a B lymphocyte or Tlymphocyte. Two major types of lymphoma are Hodgkin's lymphoma,previously known as Hodgkin's disease, and non-Hodgkin's lymphoma. Insome instance, the Non-Hodgkin lymphoma is indolent. In some instance,the Non-Hodgkin lymphoma is aggressive. Non-Hodgkin's lymphomas include,but are not limited to, diffuse large B cell lymphoma, follicularlymphoma, mucosa-associated lymphatic tissue lymphoma (MALT), small celllymphocytic lymphoma, mantle cell lymphoma, Burkitt's lymphoma,mediastinal large B cell lymphoma, Waldenstrom macroglobulinemia, nodalmarginal zone B cell lymphoma (NMZL), splenic marginal zone lymphoma(SMZL), extranodal marginal zone B cell lymphoma, intravascular large Bcell lymphoma, primary effusion lymphoma, and lymphomatoidgranulomatosis.

Methods of Administration

Provided herein are methods for treatment of a condition in a subjectcomprising administering cells disclosed herein. The methods maycomprise administering DC. The methods may comprise administering DCsafter pulsing the DCs, without storing or shipping the DCs. The methodsmay comprise administering the DCs after storing or shipping the DCs.The methods may comprise administering DCs at a time point selected fromabout 1 hour to about 24 hours after pulsing the DC. The methods maycomprise administering the DCs at a time point selected from about 1 dayto about 30 days after pulsing the DCs. The methods may compriseadministering the DCs at a time point selected from about 1 week toabout 12 weeks after pulsing the DCs.

Provided herein are methods for treatment of a condition in a subjectcomprising administering DCs to a subject in need thereof. In someinstances, the DCs are provided in a solution. In some instances, theDCs are administered by a route selected from subcutaneous injection,intramuscular injection, intradermal injection, percutaneousadministration, intravenous (“i.v.”) administration, intranasaladministration, intralymphatic injection, and oral administration. Insome embodiments, iv administration is preferable, eliciting a moredesirable response than other forms of administration (e.g. subcutaneousinjection). In some instances, the subject is infused with the DC by anintralymphatic microcatheter.

Methods described herein may comprise suspending or mixing cells in asolution for intravenous (i.v.) administration (e.g., a 0.9% NaCLsolution). The i.v. DCs may traffic to the lungs, where some will betrapped, but the majority may pass to secondary lymphatic organs such asliver and spleen white pulp T-cell zones to prime the CTL.

In some instances, the Dengue virus is initially administered at least24 hours before administering the dendritic cells. In some instances,the Dengue virus is initially administered between about 12 hours andabout 96 hours before administering the dendritic cells. In someinstances, the Dengue virus is initially administered between about 24hours and about 72 hours before administering the primed dendriticcells. In some instances, the Dengue virus is initially administeredbetween 1 day and 4 days before administering the primed dendriticcells. In some instances, the Dengue virus is administered only once. Insome instances, the Dengue virus is administered more than once. In someinstances, the Dengue virus is administered only before receivingdendritic cells. In some instances, the Dengue virus is administeredafter receiving the primed dendritic cells. In some instances, theDengue virus is administered before and after receiving the primeddendritic cells.

As seen in FIG. 2, the methods may comprise administering primed DCs onDay 0, followed by two injection of virus, such that the entiretreatment is conducted in a week or less. In some instances, the subjectwill only receive the entire treatment once. In some embodiments, theentire treatment is repeated not more than once. In some embodiments,the entire treatment is repeated not more than twice. In someembodiments, the entire treatment is repeated not more than three times.In some embodiments, the entire treatment is repeated not more than tentimes.

In some instances, the methods comprise administering the Dengue virusat a dose of about 0.5 ml of 10⁶ pfu/ml. In some instances, the dose isbetween about 10³ pfu/ml and about 10⁸ pfu/ml. In some instances, thedose is between about 10³ pfu/ml and about 10⁶ pfu/ml.

In some instances, successful infection or inoculation of the subjectwith the Dengue virus is confirmed by the development of hyperthermia orfever. In some instances, successful infection or inoculation of thesubject with the Dengue virus is confirmed by the presence or increaseof circulating cytokines in the blood/plasma of the subject. Cytokinesmay include, but are not limited to, interleukin-2 and interferon-gamma.

In some instances, methods described herein comprise administeringprimed dendritic cells to a subject in need thereof only once. In someinstances, the primed dendritic cells are administered more than once.In some instances, the primed dendritic cells are administered a firsttime and a second time, wherein the first time and the second time areseparated by about 1 day, about 2 days, about 3 days, about 4 days,about 5 days, or about 6 days, about 8 days, about 10 days, about 12days, or about 18 days. In some instances, the first time and the secondtime are separated by about 1 week, about 2 weeks, about 3 weeks, orabout a month. In some instances, the first time and the second time areseparated by more than a month. In some instances, the first time andthe second time are separated by less than 12 months. In some instances,the first time and the second time are separated by more than 12 months.

In some instances, methods described herein provide for administeringprimed dendritic cells to a subject when the subject is hyperthermic. Insome instances, primed dendritic cells are administered after thesubject has spike a fever. In some instances, primed dendritic cells areadministered after the subject's temperature has risen to between about37.5° C. and about 42° C. In some instances, the primed dendritic cellsare administered after the subject's temperature has risen to betweenabout 38° C. and about 42° C. In some instances, the primed dendriticcells are administered after the subject's temperature has risen to atleast about 38.5° C. In some instances, the primed dendritic cells areadministered after the subject's temperature has risen to 38.5° C. Insome instances, the primed dendritic cells are administered to thesubject after the subject's temperature reaches 38 degrees Celsius orhigher. In some instances, the subject's temperature is measured by atympanic or oral method.

Methods for producing primed DC described herein may comprise activatingT cells in vitro with the DCs produced by the methods disclosed herein.In some instances, the subject is incapable of mounting an effectiveimmune response. For example, the subject may be immunocompromised. Thesubject may have received a therapy that renders them immunocompromised.The subject may have a disease that renders them immunocompromised. Inthis case, the methods may comprise contacting T cells from anHLA-matched subject with the DCs. Contacting the T cells with the DCs invitro may induce a CTL response. Contacting the T cells with the DCs invitro may also induce proliferation of the T cells.

EXAMPLES Example 1. Generation and Pulsing of Murine Dendritic Cells(DC)

A method as described by Lutz M., et. al. (J. Immunol. Methods223:77-92, 1999), was employed to generate mature DCs form mouse bonemarrow. Bone marrow suspensions were incubated in petri dishes in mediumsupplemented with recombinant murine GM-CSF for 10 days. Non-adherentcells were collected, centrifuged and resuspended in medium containingGM-CSF and lipopolysaccharide. Two days later, the DCs were harvestedand their viability was determined by trypan-blue exclusion. Purity ofthe DCs was determined by flow cytometry analysis. DCs were pulsed withthe synthetic peptides at 10 μg/ml for 18 hours. After 18 hours ofincubation, DCs were harvested, washed twice in HBSS, and resuspended inHESS for additional studies (see Example 2 and 3).

Example 2. Dengue Virus and Dendritic Cells for the Treatment ofMelanoma in a First Mouse Model

A mouse model assay was performed to observe results from combinationtargeting of cancer cells using a Dengue virus (DV) strain and tumorantigen primed dendritic cells (DCs). DV C57BL/6 mice were inoculatedwith 0.05 ml of Dengue virus (DEN-2 strain #1710) at 1×10⁶ or 1×10⁷pfu/ml by injection in the base of tail. Recombinant murine IL-2(Genzyme) and IFN-gamma (Sigma Pharmaceuticals) were administered byintravenous infusion at 2,000 (rIL-2) and 500 1U (rIFN-gamma) on days 5,10, 15, and 20 following administration of Dengue virus (DEN-2 strain#1710, CDC database entry number 555, provided by Dr. Duane Gubler).Seven days after the Dengue virus administration, C57BL/6 mice wereimmunized with mouse DCs incubated with the 2 peptides separately andinjected intravenously. Peptides were synthesized. The H-2b-restrictedpeptide from Ovalbumin (OVA-8), SIINFEKL (SEQ ID NO: 7), was used as acontrol. B16 melanoma-associated H-2b-restricted peptides derived fromthe antigens gp100/pme117 (EGSRNQDWL (SEQ ID NO: 1)) and from TRP-1/75(TAYRYHLL (SEQ ID NO: 2)) were used to pulse murine DCs (see Example 1for details). Two additional immunizations with DCs were given at 14-dayintervals. Three days after the last DC infusion, mice were challengedwith 5×10⁴ viable B16 melanoma cells intravenously in the lateral tailvein and then followed for survival, which was recorded as thepercentage of surviving animals over time (in days) after tumorinjection. Data was recorded from five or more mice/group (see Table 2and FIG. 3).

TABLE 2 MOUSE NO. OF LUNG Condition Group ID METASTASES Mean DV10⁶pfu/ml + 2 × 10⁶ DCs pulsed with 2 II-2-1 55 gp100/TRP2 DV10⁶ pfu/ml + 2× 10⁶ DCs pulsed with 2 II-2-2 68 gp100/TRP2 DV10⁶ pfu/ml + 2 × 10⁶ DCspulsed with 2 II-2-3 57 gp100/TRP2 DV10⁶ pfu/ml + 2 × 10⁶ DCs pulsedwith 2 II-2-4 62 gp100/TRP2 DV10⁶ pfu/ml + 2 × 10⁶ DCs pulsed with 2II-2-5 52 58.8 gp100/TRP2 No DV + 2 × 10⁶ DCs pulsed with gp100/TRP2 1II-1-1 58 No DV + 2 × 10⁶ D DCs C pulsed with 1 II-1-2 62 gp100/TRP2 NoDV + 2 × 10⁶ DCs pulsed with gp100/TRP2 1 II-1-3 66 No DV + 2 × 10⁶ DCspulsed with gp100/TRP2 1 II-1-4 72 No DV + 2 × 10⁶ DCs pulsed withgp100/TRP2 1 II-1-5 60 63.6

The number of lung metastases observed in mice administered in Group 2(Dengue Virus serotype 2 strain #1710 and tumor peptide primed DCs) was7.5% lower than control mice in Group 1, administered the tumor peptideprimed DCs without the Dengue virus.

Example 3. Dengue Virus and Dendritic Cells for the Treatment ofMelanoma in a Second Mouse Model

A mouse model assay was performed to observe results from combinationtargeting of cancer cells using a Dengue virus (DV) strain and tumorantigen primed dendritic cells (DCs). Mice were administered cytokinesto parallel the response to DV observed in humans.

Tumors were established in mice using the H-²b-restricted B16 murinemelanoma cells line (ATCC #CRL-6322). Peptides (B16 melanoma associatedH-²b-restricted peptides derived from antigens gp100/pme117 and fromTRP-1/gp75) used for pulsing the dendritic cells were synthesized.Dendritic cells were generated from mouse bone marrow according tomethods as described in Lutz et al. (J. Immunol. Methods 223:77-92,1999).

On day 0, mice received 5×10⁴ viable B16 melanoma cells intravenously inthe lateral tail vein to establish pulmonary metastases. On day 7, themice were inoculated with 0.05 ml of Dengue virus (DEN-2 strain #1710,CDC database entry number 555) at 1×10⁶ or 1×10⁷ pfu/ml by injection inthe base of tail. Recombinant murine IL-2 (Genzyme) and IFN-gamma (SigmaPharmaceuticals) were administered by intravenous infusion at 2,000 1U(rIL-2) and 500 1U (rIFN-gamma) at 5-day intervals followingadministration of Dengue virus (DEN-2 strain #1710). On days 21, 35 and49, the mouse DCs were incubated with the 2 peptides separately andinjected intravenously in 2 sequential administrations on the same dayto match the route and schedule of administration in subjects (seeExample 2 for additional details). Control groups of mice received noDengue virus or dendritic cells pulsed with H-²b-restricted peptide fromovalbumin (OVA-8), SIINFKEL. Treatment and control groups are shown inTable 3.

TABLE 3 Dengue Virus # of dendritic cells and type of peptide Group A10⁶ pfu/ml 10⁶ DCs pulsed with gp100/pme117 (EGSRNQDWL)(SEQ ID NO: 1)10⁶ DCs pulsed with TRP-1/gp75 (TAYRYHLL)(SEQ ID NO: 2) Total 2 ×10⁶ DCs pulsed with peptide/mouse Group B 10⁶ pfu/ml10⁷ DCs pulsed with gp100/pme117 (EGSRNQDWL)(SEQ ID NO: 1)10⁷ DCs pulsed with TRP-1/gp75 (TAYRYHLL)(SEQ ID NO: 2) Total 2 ×10⁷ DCs pulsed with peptide/mouse Group C - Control None10⁶ DCs pulsed with gp100/pme117 (EGSRNQDWL)(SEQ ID NO: 1)10⁶ DCs pulsed with TRP-1/gp75 (TAYRYHLL)(SEQ ID NO: 2) Total 2 ×10⁶ DCs pulsed with peptide/mouse Group D - Control 10⁶ pfu/ml10⁶ DCs pulsed with OVA (SIINFEKL)(SEQ ID NO: 7)10⁶ DCs pulsed with OVA (SIINFEKL)(SEQ ID NO: 7) Total 2 ×10⁶ DCs pulsed with peptide/mouse

On day 90, animals were sacrificed and lung tumor colonies were counted.Pulmonary metastases were enumerated in a blinded, coded fashion afterinsufflation and fixation of the lungs with Fekette's solution. Datawere reported as the mean number of metastases; four mice/group (seeTable 4 and FIG. 4). Histopathology of the following major organ systemswere performed: brain, heart, lungs, liver, kidneys, spleen and gonads(data not shown).

TABLE 4 NO. OF LUNG Condition Group MOUSE ID METASTASES Mean DV10⁶pfu/ml + 2 × 10⁶ DC pulsed A III-1-1 82 with gp100/TRP2 DV10⁶ pfu/ml + 2× 10⁶ DC pulsed A III-1-2 87 with gp100/TRP2 DV10⁶ pfu/ml + 2 × 10⁶ DCpulsed A III-1-3 78 with gp100/TRP2 DV10⁶ pfu/ml + 2 × 10⁶ DC pulsed AIII-1-4 72 with gp100/TRP2 79.75 DV10⁷ pfu/ml + 2 × 10⁶ DC pulsed BIII-2-1 87 with gp100/TRP2 DV10⁶ pfu/ml + 2 × 10⁶ DC pulsed B III-2-2 77with gp100/TRP2 DV10⁶ pfu/ml + 2 × 10⁶ DC pulsed B III-2-3 92 withgp100/TRP2 DV10⁶ pfu/ml + 2 × 10⁶ DC pulsed B III-2-4 85 with gp100/TRP285.25 No dengue virus + 2 × 10⁶ DC C III-3-1 97 pulsed with gp100/TRP2No dengue virus + 2 × 10⁶ DC C III-3-2 94 pulsed with gp100/TRP2 Nodengue virus + 2 × 10⁶ DC C III-3-3 88 pulsed with gp100/TRP2 No denguevirus + 2 × 10⁶ DC C III-3-4 91 pulsed with gp100/TRP2 92.5 DV10⁶pfu/ml + 2 × 10⁶ DC pulsed D III-4-1 180 with OV DV10⁶ pfu/ml + 2 × 10⁶DC pulsed D III-4-2 174 with OV DV10⁶ pfu/ml + 2 × 10⁶ DC pulsed DIII-4-3 165 with OV DV10⁶ pfu/ml + 2 × 10⁶ DC pulsed D III-4-4 177 withOV 174

The number of lung metastases observed in mice in Group C (administeredtumor antigen primed DCs and no virus) was 47% less than control Group D(administered DENV-2 #1710 and DCs exposed to a control peptide). Thenumber of lung metastases observed in mice in Group A (administeredDENV-2 #1710 and tumor antigen primed DCs) was 54% less than controlGroup D (administered DENV-2 #1710 and DCs exposed to a controlpeptide). The number of lung metastases observed in mice in Group B(administered DENV-2 #1710 and tumor antigen primed DCs) was 51% lessthan control Group D (administered DENV-2 #1710 and DCs exposed to acontrol peptide). The average reduction in Group A and B compared toGroup D was 52.8%.

Example 4. Manufacture and Screening of Less-Pathogenic Dengue Virus

A Master Cell Bank with validated and certified cell lines from Vero(African Green Monkey Kidney Cells) was generated and tested for absenceof any contaminants and adventitious organisms. Vero lines are used bythe World Health Organizations to produce a variety of viral vaccines.Dengue virus was passaged in a validated Vero Line derived from theMaster Cell Bank and established as a Working Cell Bank according toguidelines established by the FDA Center for Biologics (CBER). TwoDengue Virus Type 2 strains (DNV-2 #1584 and DENV-2 #1710) from initialseed stocks were added to the Vero Cells of the WCB at a MOI of 10⁻⁵.

The first 4-ml overlay medium—containing 1% SeaKem LE agarose (FMCBioProducts, Rockland, Me.) in nutrient medium (0.165% lactalbuminhydrolysate [Difco Laboratories, Detroit, Mich.]), 0.033% yeast extract[Difco], Earle's balanced salt solution, 25 mg of gentamicin sulfate[BioWhittaker, Walkersville, Md.] and 1.0 mg of amphotericin B[Fungizone; E. R. Squibb & Sons, Princeton, N.J.], per liter and 2%FBS)—was added after adsorption of the 200-ml virus inoculum for 1.5 hat 37° C. Following incubation at 37° C. for 7 days, a second 2-mloverlay containing additional 80 mg of neutral red vital stain(GIBCO-BRL, Gaithersburg, Md.) per ml was added. Plaques were counted 8to 11 days after infection.

A plaque assay on final virus cultures was performed. The titer of DNV-2#1584 was about 5E+06 PFU/ml, and the titer of DENV-2 #1710 was 3.5E+06pfu/mL as estimated from plaque assays. Dengue virus 2 (DNV-2; #1584)from ATCC showed a clear cytopathic effect in Vero cells 5 days postinfection, whereas Vero cells appears to have a morphology change 11days post infection of the blind passage #2 (#1710 virus). (Data notshown.) The assay shows that the DENV-2 #1710 virus is far lesscytopathic than the DNV-2 #1584 strain

Example 5. Cancer Killing Assay with Pulsed DC, with and without DV

In a control arm, normal human tumor infiltrating lymphocytes (TILs)were directly applied to human melanoma FEMX cells. T-cell receptorswere matched to FEMX melanoma cell line via HLA A2.1+. In a treatmentarm human TILs were exposed to DV supernatants containing interferonsand interleukins. Exposed TILs+DV supernatants were placed in culturewith FEMX tumor cells. Both arms were left to kill cancer cells for 4hours at a ratio of 5-to-1T-cell to tumor cell (100,000 cells to 20,000cells). Surviving tumor cells were then counted as % of starting cellsby flow cytometry. Results, shown in Table 5, demonstrate that DVinduces 35% additional cancer cell killing beyond the pulsed DCanti-cancer response.

TABLE 5 DV enhancement of pulsed DC anti-cancer activity % FL2-A− %FL2-A+ (% Apoptotic Cells) CTL 86.1% 13.9% CTL + DV Sups 81.2% 18.8%

Example 6. Human Dendritic Cell Isolation and Pulse with Melanoma LysateAntigens

The following example demonstrates generation of a highly pure CD11a+mature DC population expressing high levels of human IL-12p70 from pure,isolated CD14+ monocytes, as well as priming of the DC with melanomacell lysate, the entire process being completed in less than one week.Cells were cultured on hard plastic plates and not exposed to softplastic bags.

CD14+ monocytes were isolated and analyzed for expression of CD14, CD15,CD45 and 7AAD. Post-prodigy run, 90.25% of input cells were CD14+(seeFIG. 5). CD14+ cells were treated with GM-CSF and IL-4 24 hours postplating to generate immature dendritic cells.

RPMI-7951 melanoma cells from ATCC arrived on the day of the prodigy runand were re-suspended, counted and plated. Melanoma cells were thantreated with a calcium hypochlorite solution. Alternatively, cells weretreated with sodium chlorite solution. The melanoma cell lysate wasadded to the immature DC, and maturing agents IFN gamma (1000U/mL), R848(5 μg/mL) and LPS (long/mL) were added.

Supernatant from mature DCs were collected and examined for mycoplasmaand endotoxin 22 hours after pulsing with melanoma cell lysate and 18hours after addition of maturing agents. No pathogenic organisms wereobserved. An ELISA assay was performed to measure IL-12p70 levels, usingsupernatant from the DC culture medium. The concentration of IL-12p70was 19+/−4 ng/mL in a first batch of cells, as opposed to the industrystandard of 4-6 ng/mL. Various batches of cells produced by the sameprotocol showed concentrations of 15-23 ng/mL IL-12p70. A viabilityassay was conducted and average viability was recorded at 79.2% aftercells were frozen, thawed and cultured (compared to a comparator whichshowed 70%). FIG. 6 shows DC IL-12p70 production (see Sample 1 of FIG.6) relative to that of several comparators. These comparators methodsinclude exposing cells to soft plastic bags, lysing cells with solutionsother than a chlorite solution, and do not use the combination of LPS,IFN gamma and R848 to mature cells.

Cells were further frozen and then thawed at 4° C. to test cell countsand viability after freezing and thawing. These were measured atapproximately 16 h, 18 h, 20 h and 22 h after beginning of thaw. SeeTable 6 for results. An extra harvest of non-pulsed DCs were tested in acryopreservation study, and showed greater than 70% viability.Pre-cryopreservation viability ranged from 85-89%.

TABLE 6 Pulsed DC viability after freeze-thaw Time post start thawViability Total Live Cells in 30 ml 16 h 68.9% 8.52 × 10{circumflex over( )}6 18 h 67.9% 8.25 × 10{circumflex over ( )}6 20 h 66.3% 7.23 ×10{circumflex over ( )}6 22 h 70.3% 11.46 × 10{circumflex over ( )}6 

Switch from these slow thaws (16 h to 22 h, shown in Table 6) to a rapidthaw (37° C. water bath for about 30 sec to about 5 minutes), resultedin viability from 71-79%.

Cells not pulsed were used as a cryopreservation study. Viability rangedfrom 71.4% to 79.2%.

Example 7. Inducing Cytokines in Human White Blood Cells with DengueVirus

Human white blood cells (WBC), including monocytes, dendritic cells andT lymphocytes, were infected with either mock virus or Dengue virus atthree different multiplicities of infection (MOD, MOI of 0.1, MOI of 0.5and MOI of 2 at time=0. Levels (pg/mL) of various cytokines weremeasured at 48 h, 72 h and 96 h, post-infection. Treatments wereperformed in triplicate. Results are shown for each time point in Tables7-9. (M=mock. 0.1, 0.5 and 2 are MOI). Triplicate average of changesbetween mock and Dengue virus at the assessed MOIs was calculated andshown as a percentage in Table 10.

TABLE 7 Cytokine levels produced by human WBC, 48 h post- Dengue virusinfection M M M 0.1 0.1 0.1 IL-1b 15 6 6 6 6 6 IL-10 4 4 4 4 4 4 IL-1311 11 11 11 11 11 IL-6 12 7 9 941 874 788 IL-12 19 12 13 14 15 15 Rantes12 11 11 14 16 18 CCL- 3 3 3 3 3 3 11 IL-17 18 18 18 18 18 18 MIP- 123110 109 183 166 219 1a GM- 5 5 5 5 5 5 CSF MIP- 83 78 82 123 111 118 1bMCP-1 1.77e+03 1.48e+03 1.87e+03 12.6e+03 10.4e+03 9.95e+03 IL-15 33 3333 33 33 33 IL-5 8 8 8 8 8 8 IFN-g 5 5 5 6 6 6 IFN-a 16 12 12 37 35 33IL- 3.37e+03 2.84e+03 3.59e+03 4.99e+03 4.39e+03 4.30e+03 1Ra TNF-a 6 66 8 8 8 IL-2 9 9 9 9 9 9 IL-7 16 8 11 31 27 26 IP-10 4 4 4 23 15 18IL-2R 31 31 31 54 47 52 MIG 38 32 39 29 26 26 IL-4 23 23 23 23 23 23IL-8 17.8e+03 17.8e+03 17.8e+03 17.8e+03 17.8e+03 17.8e+03 0.5 0.5 0.5 22 2 IL-1b 6 6 6 7 7 7 IL-10 4 4 5 5 4 4 IL-13 11 11 11 11 11 11 IL-68.08e+03 8.64e+03 10.0e+03 11.2e+03 11.2e+03 11.2e+03 IL-12 17 20 19 2825 25 Rantes 32 56 64 152 135 148 CCL- 3 3 3 3 3 3 11 IL-17 18 18 18 1818 18 MIP- 212 309 328 261 264 259 1a GM- 5 6 7 22 20 21 CSF MIP- 145152 142 163 149 155 1b MCP-1 21.8e+03 23.4e+03 24.2e+03 32.0e+0332.0e+03 32.0e+03 IL-15 33 33 33 68 63 60 IL-5 16 18 18 21 21 20 IFN-g 88 8 10 9 10 IFN-a 47 50 47 67 68 71 IL- 4.55e+03 4.88e+03 5.14e+034.13e+03 3.42e+03 3.82e+03 1Ra TNF-a 16 13 11 21 21 19 IL-2 9 9 9 9 9 9IL-7 51 49 47 53 55 54 IP-10 39 46 39 218 128 147 IL-2R 57 69 69 79 7679 MIG 26 31 28 23 22 27 IL-4 27 27 27 30 29 30 IL-8 17.8e+03 17.8e+0317.8e+03 17.8e+03 17.8e+03 17.8e+03

TABLE 8 Cytokine levels produced by human WBC, 72 h post- Dengue virusinfection M M M 0.1 0.1 0.1 IL-1b 6 6 6 6 6 6 IL-10 4 4 4 4 4 4 IL-13 1111 11 11 11 11 IL-6 7 7 7 637 690 737 IL-12 12 11 11 12 12 14 Rantes 1111 11 11 11 11 CCL- 3 3 3 3 3 3 11 IL-17 18 18 18 18 18 18 MIP- 96 88 8884 97 118 1a GM- 5 5 5 5 5 5 CSF MIP- 83 78 80 85 90 101 1b MCP-15.51e+03 5.02e+03 4.87e+03 21.5e+03 22.4e+03 21.7e+03 IL-15 33 33 33 3333 33 IL-5 8 8 8 8 8 8 IFN-g 5 5 5 6 6 6 IFN-a 26 23 24 43 46 46 IL-6.30e+03 5.97e+03 6.02e+03 6.36e+03 6.89e+03 6.36e+03 1Ra TNF-a 6 6 6 66 6 IL-2 9 9 9 9 9 9 IL-7 8 8 8 23 25 21 IP-10 4 4 4 18 14 17 IL-2R 3128 20 42 44 42 MIG 40 35 35 32 28 27 IL-4 23 23 23 23 23 23 IL-817.8e+03 17.8e+03 17.8e+03 17.8e+03 17.8e+03 17.8e+03 0.5 0.5 0.5 2 2 2IL-1b 6 6 6 6 7 7 IL-10 5 5 4 4 5 5 IL-13 11 11 11 11 11 11 IL-6 55188803 6841 11.2e+03 11.2e+03 11.2e+03 IL-12 15 17 16 17 20 22 Rantes 1116 15 21 88 68 CCL- 3 3 3 3 3 3 11 IL-17 18 18 18 18 18 18 MIP- 91 118106 54 133 87 1a GM- 5 5 5 8 15 15 CSF MIP- 104 112 101 84 98 101 1bMCP-1 32.0e+03 32.0e+03 32.0e+03 32.0e+03 32.0e+03 32.0e+03 IL-15 33 3333 33 38 67 IL-5 14 15 14 17 19 20 IFN-g 8 8 7 6 8 8 IFN-a 62 56 52 6166 67 IL- 6.90e+03 6.76e+03 6.01e+03 4.33e+03 3.89e+03 4.39e+03 1RaTNF-a 6 6 6 6 6 6 IL-2 9 9 9 9 9 9 IL-7 42 40 40 45 50 48 IP-10 42 38 38104 143 169 IL-2R 42 47 47 44 56 60 MIG 27 25 22 24 19 25 IL-4 27 25 2426 27 29 IL-8 17.8e+03 17.8e+03 17.8e+03 17.8e+03 17.8e+03 17.8e+03

TABLE 9 Cytokine levels produced by human WBC, 96 h post- Dengue virusinfection M M M 0.1 0.1 0.1 IL-1b 6 6 6 6 6 6 IL-10 4 4 4 5 4 4 IL-13 1111 11 11 11 11 IL-6 9 9 9 834 734 771 IL-12 14 13 13 16 14 14 Rantes 1111 11 11 11 11 CCL- 3 3 3 3 3 3 11 IL-17 18 18 18 18 18 18 MIP- 98 89119 73 103 122 1a GM- 5 5 5 5 5 5 CSF MIP- 82 78 99 63 89 99 1b MCP-18.19e+03 7.61e+03 7.10e+03 32.0e+03 25.3e+03 25.6e+03 IL-15 33 33 33 3333 33 IL-5 8 8 8 8 8 8 IFN-g 6 6 7 8 7 6 IFN-a 27 29 27 52 47 44 IL-10.9e+03 10.9e+03 10.2e+03 11.0e+03 9.57e+03 9.56e+03 1Ra TNF-a 6 6 6 66 6 IL-2 9 9 9 9 9 9 IL-7 8 8 8 21 18 14 IP-10 4 4 4 29 11 11 IL-2R 2523 28 39 36 42 MIG 39 40 39 39 24 26 IL-4 23 23 23 23 23 23 IL-817.8e+03 17.8e+03 17.8e+03 17.8e+03 17.8e+03 17.8e+03 0.5 0.5 0.5 2 2 2IL-1b 6 6 7 7 6 7 IL-10 5 6 6 5 5 5 IL-13 11 11 11 11 11 11 IL-6 70267.47e+03 7.65e+03 11.2e+03 11.2e+03 11.2e+03 IL-12 16 14 16 16 20 20Rantes 11 11 11 37 70 68 CCL- 3 3 3 3 3 3 11 IL-17 18 18 18 18 18 18MIP- 79 77 85 60 108 106 1a GM- 5 5 5 12 14 15 CSF MIP- 85 83 89 67 7276 1b MCP-1 32.0e+03 32.0e+03 32.0e+03 32.0e+03 32.0e+03 32.0e+03 IL-1533 33 33 49 43 52 IL-5 15 16 16 20 19 18 IFN-g 7 7 7 7 7 7 IFN-a 56 5865 64 64 67 IL- 7.63e+03 7.80e+03 8.27e+03 5.49e+03 4.22e+03 4.45e+031Ra TNF-a 6 6 6 6 6 6 IL-2 9 9 9 9 9 9 IL-7 33 37 48 50 45 44 IP-10 2928 33 134 101 104 IL-2R 39 42 59 52 49 57 MIG 19 22 24 20 17 18 IL-4 2524 25 27 27 28 IL-8 17.8e+03 17.8e+03 17.8e+03 17.8e+03 17.8e+0317.8e+03

TABLE 10 Relative changes in WBC cytokine levels between mock and Dengueinfections MOI 0.1 MOI 0.5 MOI 2 48 h 72 h 96 h 48 h 72 h 96 h 48 h 72 h96 h IL-1b −33% 0% 0% −33% 0% 6% −22% 11% 11% IL-10 0% 0% 8% 8% 17% 42%8% 17% 25% IL-13 0% 0% 0% 0% 0% 0% 0% 0% 0% IL-6 9.20E+03% 9.73E+03%8.56E+03% 95.4E+03% 10.1E+04% 8.19E+03% 12.02E+04% 16.04E+04% 12.46E+04%IL-12 0% 12% 10% 27% 41% 15% 77% 74% 40% Rantes 41% 0% 0% 347% 27% 0%1179% 436% 430% CCL- 0% 0% 0% 0% 0% 0% 0% 0% 0% 11 IL-17 0% 0% 0% 0% 0%0% 0% 0% 0% MIP- 66% 10% −3% 148% 16% −21% 129% 1% −10% 1a GM- 0% 0% 0%20% 0% 0% 320% 153% 173% CSF MIP- 45% 15% −3% 81% 32% −1% 92% 17% −17%1b MCP-1 543% 325% 262% 1255% 523% 319% 1774% 523% 319% IL-15 0% 0% 0%0% 0% 0% 93% 39% 45% IL-5 0% 0% 0% 117% 79% 96% 158% 133% 138% IFN-g 20%20% 11% 60% 53% 11% 93% 47% 11% IFN-a 163% 85% 72% 260% 133% 116% 415%166% 135% IL- 39% 7% −6% 49% 7% −26% 16% −31% −56% 1Ra TNF-a 33% 0% 0%122% 0% 0% 239% 0% 0% IL-2 0% 0% 0% 0% 0% 0% 0% 0% 0% IL-7 140% 188%121% 320% 408% 392% 363% 496% 479% IP-10 367% 308% 325% 933% 883% 650%4008% 3367% 2725% IL-2R 65% 62% 54% 110% 72% 84% 152% 103% 108% MIG −26%−21% −25% −22% −33% −45% −34% −38% −53% IL-4 0% 0% 0% 17% 10% 7% 29% 19%19% IL-8 0% 0% 0% 0% 0% 0% 0% 0% 0%

Example 8. Additional Virus Manufacturing Protocols

In addition to methods of Example 4, both Vero and FRhL cells areinfected using dilutions of the supernatant from blind passage #2,DENV-2 #1710 and DNV-2 #1584, respectively. In order to increase thedetection sensitivity, an immunofluorescence staining is developed todetect virus in the cells infected with supernatant from blind passage#2.

Ultracentrifugation is used to concentrate virus when necessary.Following confirmation of virus titer, final product is filtered toremove any cellular debris, assessed for absence of any adventitiousorganisms, and upon final lot released, bottled in 5 ml bottles, andstored at 4° C. until ready for shipment and administration.

Example 9. Collection of PBMC from Donors

Donors (either autologous or HLA-matched allogenic) have a leukapheresisprocedure performed at a facility with trained personnel and properequipment. After the apheresis is complete, the red cells, platelets,and plasma proteins are returned to the donor. The apheresis product isassessed at the site (Gram Stain test and Limulus Amoeba Lysis [LAL])for presence of bacterial contamination. After passing, the collectioncontainer (with small testing sample container attached), is barcodedwith donor-specific information and placed in an approved shippingcontainer conforming to both FDA and DOT regulations for storage andshipping of non-infectious biological materials. The shipping containeris packaged with a cooling element (e.g., solid CO₂, Liquid N₂), andtemperature monitors. The shipping container is a hard plastic flask. Acourier transports the container within 24 hours to the GMPmanufacturing facility.

Example 10. Manufacture and Use of Dendritic Cells Pulsed with TumorAntigens

Monocytes are separated from other collected white blood cells (e.g. Tcells. B cells, NK cells, eosinophils and basophils). This isaccomplished with immuno-magnetic selection or, alternatively, byadherence properties. Immuno-magnetic selection involves pouring thewhite blood cells into a sterile plastic column with plastic beadscoated with antibodies for immune cell CD surface proteins:(CD4/CD8/CD56, etc.).

An example of immunomagnetic selection is the EasySep MonocyteEnrichment kit available from Stem Cell Technologies (Vancouver, B.C,Canada, www.stemcell.com). To use the EasySep kit, the apheresis productis suspended in sterile PBS and poured into the EasySep plastic columncontaining Tetrameric antibody complexes with murine antibodies for:human CD2, CD3, CD16, CD19, CD20, CD56, CD66b, CD123, and Glycophorin A.After incubation for 10 minutes, EasySep magnetic particles are added.The cells adhering to the beads removed an electromagnet sorting. Themagnet is inverted, and the desired cell fraction (monocytes), is pouredinto a sterile polystyrene flask for additional processing. Alternately,in a positive adherence selection assay, magnetic beads coated withCD1+/CD14+ antibodies is mixed with monocytes, a magnet is placedagainst the column, and non-binding cells are flushed out of the columnwith PBS solution. The monocytes are then washed off the beads. Inpositive adherence selection, the properties of monocytes to stick tocertain surfaces are used to separate them by running the apheresisproduct down a slanted column.

Alternatively, bone marrow cells are depleted for lymphocytes and MHCClass positive cells by Fluorescent Activated Cell Sorting (FACS) withmonoclonal antibodies for CD3, CD4, and CD8. Remaining cells arecultured overnight at 37° C. in a 5% CO₂ atmosphere in a basal cellculture medium supplemented with human AB serum. Human AB serum ischosen because it grows cells at a faster rate than other serum types,and serum free media produces DCs with much lower T-cell stimulationcapability. After 24 hours, the cells are replated and cultured in thepresence of Granulocyte-Macrophage Colony Stimulation Factor (GM-CSF),and recombinant IL-4 at 900 U/ml. After 3 to 4 days, media to beexchanged for fresh cytokine media.

Alternatively, dermal dendritic cells (DDCs) are prepared using thefollowing methods: Keratomes from healthy human volunteers are incubatedin a solution of the bacterial proteases Dispase type 2 at a finalconcentration of 1.2 U/ml in RPMI 1640 for 1 hour at 37° C. After theincubation period, epidermis and dermis are easily separated. Epidermaland dermal sheets are then cut into small (1-10 mm) pieces after severalwashing with PBS, and placed in RPMI 1640 supplemented with 10% FetalBovine Serum (FBS), and placed in 10-cm tissue culture plates. After 2-3days, pieces of tissue are removed, and the medium collected. Cellsmigrating out of the tissue sections into the medium are spun down,resuspended in 1-2 ml fresh medium and stained with trypan blue. Furtherenrichment is achieved by separation on a metrizamide gradient. Cellsare layered onto 3-ml columns of hypertonic 14.5% metrizamide andsedimented at 650 g for 10 minutes at room temperature. Low densityinterphase cells are collected and washed in two successively lesshypertonic washes (RPMI 1640 with 10% FBS and 40 mM NaCl) to returncells to isotonicity.

When the monocytes are collected, they may number only a few thousand.The recombinant human growth factors rhuInterleukin-4 (IL-4), andrhuGranulocyte-Macrophage-Colony-Stimulation Factor (GM-CSF), are usedin a multi-step protocol to accomplish the expansion of DC numbers tothe range of 50 million. After the addition of IL-4 and GM-CSF, cellsare assessed for and expansion in number and the development ofmature-DC markers: (CD11⁺, CD80⁺, CD83⁺), as well as increasedexpression of both Class I (for presentation of short peptides to CD8⁺,and Class II MHC complexes (for presentation of longer peptides to CD4⁺Helper-Inducer T lymphocytes). After approximately 3-4 days, the numberof mature DCs will be measured. For example, the monocyte-enrichedfraction is placed in Nuclon-coated Cell Factory (Thermoscientific),with serum-free DC media (CellGro, Inc.), supplemented with GMP-2% humanAB serum, 500 IU/ml (about 50 ng/ml) rhuIL-4 (CellGenix), with 500 IU/ml(about 50 ng/ml) rhuGM-CSF (CellGenix), added after the first 24 hours.Final product is approximately about 1 L of total media volume. Afterabout 72 hours of culture, a population of immature DCs are assessed forthe following markers: CD1⁺ CD11⁺ CD14⁺.

Example 11. Pulsing the Dendritic Cells

A variety of tumor antigen sources are used for high-quality DCs:peptides, lysate from autologous tumors, whole tumor cells, and RNAcoding for specific tumor antigens. An excisional biopsy or blood samplecontaining leukemic or lymphoma cells are obtained by surgery or blooddraw followed by a magnetic selection to obtain leukemia/lymphoma cells.Once the tumor cells are obtained, they are barcoded and shipped inapproved containers similar to those described for apheresis previouslyto the GMP facility. Samples may be frozen at −70° C. after passingbacterial contamination tests.

Whole autologous tumor cell lysate is prepared by several methods. Toprepare the lysate, the tumor sample may be rewarmed to approximately35° C. using a water bath or other procedure. The development ofautomated cell processors like the Miltenyi GentleMACS system allows thesample to be manually minced, suspended in PBS solution, then apre-selected tissue-specific software-controlled rotor system separatesthe tumor cells. Cells are added to an enzyme mixture before beingtransferred to the Miltenyi GentleMACS dissociator. The single-cellsuspension can be membrane-lysed with minimal damage to tumor peptides,using a hypochlorite solution, which will kill any residual tumor cells,neutralize dT_(H)2 cytokines, and increase immunogenicity for superiorCTL affinity, avidity and activation. After adding hypochlorite, cultureplates are incubated at 37 degrees Celsius, 5% CO₂, for 1 hour, withgentle manual agitation at 30 min to disperse hypochlorite. Cells arewashed two time to neutralize the lysis reaction (e.g. with HBSS).Hypochlorite-treated cells may be subjected to subsequent freeze-thawcycles. Alternatively, the sample does not separate the tumor cells.Instead the sample is left to contain tumor cells and supporting cells(e.g. cells from the tumor microenvironment). Cells are lysed withcalcium hypochlorite to eliminate red blood cells and produce apoptoticand necrotic bodies without destroying peptides needed for CTLinduction.

Lysate from the GentleMACS is added on the third day of immature DCsproduction. Immature DCs are co-cultured with tumor lysate for about 16hours. The final step is maturation with an inflammatory signal.Clinical-Grade LPS (60 EU/ml) (R & D Invivogen), and Interferon-gamma(2000 IU/ml, about 100 ng/ml) (R&D Systems) are added to the flask andincubated for about 12 hours to mature the pulsed DC. After exposure toLPS, the DCs are assessed for up-regulation of CD80/CD83⁺ activationmarkers, and increase production of IL-12p70. In process testing at thisstage includes sterility (as previously described), viability (% viablecells by Trypan Blue dye exclusion), and specificity (% DC measured byCD11c flow cytometry).

After final sterility, specificity, and viability assessment, the DCsare transferred to hard plastic containers suitable for freezing at−70′C in liquid N₂, storage up to 1 year, and shipping to the clinic foruse. The containers are shipped cool overnight, then re-warmed to 37° C.in a warm-water bath before intravenous administration with a 0.9% NaCLsolution concurrent over 30 minutes. The DCs are administeredintravenously.

Example 12. Combination Delivery for Treatment of Cancer

Administration of the Dengue Virus is similar to that of other viralvaccine injections. A subject has an area of skin in the shoulder(deltoid) region cleaned with alcohol, then 0.5 ml of the virus isinjected under the skin to mimic a mosquito bite. Once the subject has afever the reaches 38.5° C., after 2-3 days from DV injection, thesubject is infused by intralymphatic microcatheter with pulsed (primed)dendritic cells. Injections are repeated until the subject is negativefor disease. DC fusions will use cells as manufactured in Example 6.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A method for treating or reducing melanoma,comprising: obtaining primed dendritic cells by a process comprising thesteps of: contacting dendritic cells with a tumor antigen,lipopolysaccharide, R848, and interferon-gamma; administering the primeddendritic cells to a subject in need thereof; and administering a DengueVirus to the subject, wherein the Dengue Virus is serotype 2 strain#1710, and wherein the subject has melanoma.
 2. The method of claim 1,wherein the primed dendritic cells are administered intravenously. 3.The method of claim 1, wherein the tumor antigen is obtained from acancer cell of the subject.
 4. The method of claim 3, wherein the primeddendritic cells are autologous or allogenic to the subject.
 5. Themethod of claim 1, wherein the primed dendritic cells produce 15 to 19ng/mL of IL-12p70.
 6. The method of claim 1, wherein the primeddendritic cells produce 15 to 23 ng/mL of IL-12p70.
 7. The method ofclaim 1, wherein the subject is a human.
 8. The method of claim 1,wherein the subject is a mouse.
 9. The method of claim 1, wherein theprimed dendritic cells are prepared by culturing on a hard surface. 10.The method of claim 9, wherein the hard surface is substantially freefrom a fluorinated polyethylene, a fluorinated polypropylene, or aphthalate.
 11. The method of claim 1, wherein the Dengue Virus isisolated from a Vero cell line.